MALDI-TOF MS: An alternative approach for ribotyping Clostridioides difficile isolates in Brazil.

Clostridioides difficile is an important organism causing healthcare-associated infections. It has been documented that specific strains caused multiple outbreaks globally, and patients infected with those strains are more likely to develop severe C. difficile infection (CDI). With the appearance of a variant strain, BI/NAP1 ribotype 027, responsible for several outbreaks and high mortality rates worldwide, the epidemiology of the CDI changed drastically in the United States, Europe, and some Latin American countries. Although the epidemic strain 027 was not yet detected in Brazil, there are ribotypes exclusively found in the country, such as, 131, 132, 133, 135, 142 and 143, which are responsible for outbreaks in Brazilian hospitals and nursing homes. Although PCR-ribotyping is the most used method in epidemiology studies of C. difficile, it is not available in Brazil. This study aimed to develop and validate an in-house database for detecting C. difficile ribotypes, usually involved in CDI in Brazilian hospitals, by using MALDI-TOF MS. A database with 19 different ribotypes, 13 with worldwide circulation and 6 Brazilian-restricted, was created based on 27 spectra readings of each ribotype. After BioNumerics analysis, neighbor-joining trees revealed that spectra were distributed in clusters according to ribotypes, showing that MALDI-TOF MS could discriminate all 19 ribotypes. Moreover, each ribotype showed a different profile with 42 biomarkers detected in total. Based on their intensity and occurrence, 13 biomarkers were chosen to compose ribotype-specific profiles, and in silico analysis showed that most of these biomarkers were uncharacterized proteins or well-conserved peptides, such as ribosomal proteins. A double-blind assessment using the 13 biomarkers correctly assigned the ribotype in 73% of the spectra analyzed, with 94%-100% of correct hits for 027 and for Brazilian ribotypes. Although further analyses are required, our results show that MALDI-TOF MS might be a reliable, fast and feasible alternative for epidemiological surveillance of C. difficile in Brazil.

Intracellular changes of a swine tracheal cell line infected with a Mycoplasma hyopneumoniae pathogenic strain.

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), a widespread disease that causes major economic losses to the pig industry. The swine host response plays an important role in the outcome of M. hyopneumoniae infections. The whole proteome of newborn pig trachea (NPTr) epithelial cells infected with the M. hyopneumoniae pathogenic strain 7448 was analyzed using an LC-MS/MS approach to shed light on intracellular processes triggered in response to the pathogen. Overall, 853 swine protein species were identified, 156 of which were differentially represented in response to M. hyopneumoniae 7448 infection in comparison with non-infected control cells. These differentially represented proteins were categorized by function. Fifty-seven of them were assigned to the immune system and/or response to stimulus functional subcategories. Comparative expression analysis of these immune-related proteins in NPTr cells infected with attenuated or non-pathogenic mycoplasmas (M. hyopneumoniae J strain and M. flocculare, respectively) revealed proteins whose abundance was altered only in response to the pathogenic M. hyopneumoniae 7448 strain. Among these proteins, calcium homeostasis and endoplasmic reticulum stress-related biomarkers were detected, providing evidence of molecular mechanisms that might lead to swine cell apoptosis.

Vagococcus bubulae sp. nov., isolated from ground beef, and Vagococcus vulneris sp. nov., isolated from a human foot wound.

Two unusual catalase-negative, Gram-stain-positive, Vagococcus-like isolates that were referred to the CDC Streptococcus Laboratory for identification are described. Strain SS1994T was isolated from ground beef and strain SS1995T was isolated from a human foot wound. Comparative 16S rRNA gene sequence analysis of isolates SS1994T and SS1995T against Vagococcus type strain sequences supported their inclusion in the genus Vagococcus. Strain SS1994T showed high sequence similarity (>97.0 %) to the two most recently proposed species, Vagococcus martis (99.2 %) and Vagococcus teuberi (99.0 %) followed by Vagococcus penaei (98.8 %), strain SS1995T (98.6 %), Vagococcus carniphilus (98.0 %), Vagococcus acidifermentans (98.0 %) and Vagococcus fluvialis (97.9 %). The 16S rRNA gene sequence of strain SS1995T was most similar to V. penaei (99.1 %), followed by SS1994T (98.6 %), V. martis (98.4 %), V. teuberi (98.1 %), V. acidifermentans (97.8 %), and both V. carniphilus and V. fluvialis (97.5 %). A polyphasic taxonomic study using conventional biochemical and the rapid ID 32 STREP system, MALDI-TOF MS, cell fatty acid analysis, pairwise sequence comparisons of the 16S rRNA, rpoA, rpoB, pheS and groL genes, and comparative core and whole genome sequence analyses revealed that strains SS1994T and SS1995T were two novel Vagococcus species. The novel taxonomic status of the two isolates was confirmed with core genome phylogeny, average nucleotide identity <84 % and in silico DNA-DNA hybridization <28 % to any other Vagococcus species. The names Vagococcusbubulae SS1994T=(CCUG 70831T=LMG 30164T) and Vagococcusvulneris SS1995T=(CCUG 70832T=LMG 30165T) are proposed.

Accurate and selective quantification of anthrax protective antigen in plasma by immunocapture and isotope dilution mass spectrometry.

Anthrax protective antigen (83 kDa, PA83) is an essential component of two major binary toxins produced by Bacillus anthracis, lethal toxin (LTx) and edema toxin (ETx). During infection, LTx and ETx contribute to immune collapse, endothelial dysfunction, hemorrhage and high mortality. Following protease cleavage on cell receptors or in circulation, the 20 kDa (PA20) N-terminus is released, activating the 63 kDa (PA63) form which binds lethal factor (LF) and edema factor (EF), facilitating their entry into their cellular targets. Several ELISA-based PA methods previously developed are primarily qualitative or semi-quantitative. Here, we combined protein immunocapture, tryptic digestion and isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS), to develop a highly selective and sensitive method for detection and accurate quantification of total-PA (PA83 + PA63) and PA83. Two tryptic peptides in the 63 kDa region measure total-PA and three in the 20 kDa region measure PA83 alone. Detection limits range from 1.3-2.9 ng mL-1 PA in 100 µL of plasma. Spiked recovery experiments with combinations of PA83, PA63, LF and EF in plasma showed that PA63 and PA83 were quantified accurately against the PA83 standard and that LF and EF did not interfere with accuracy. Applied to a study of inhalation anthrax in rhesus macaques, total-PA suggested triphasic kinetics, similar to that previously observed for LF and EF. This study is the first to report circulating PA83 in inhalation anthrax, typically at less than 4% of the levels of PA63, providing the first evidence that activated PA63 is the primary form of PA throughout infection.

Messenger RNA levels of the Polo-like kinase gene (PLK) correlate with cytokinesis in the Trypanosoma rangeli cell cycle.

BACKGROUND: Trypanosoma rangeli is a protozoan parasite that is non-virulent to the mammalian host and is morphologically and genomically related to Trypanosoma cruzi, whose proliferation within the mammalian host is controversially discussed. OBJECTIVES: We aimed to investigate the T. rangeli cell cycle in vitro and in vivo by characterizing the timespan of the parasite life cycle and by proposing a molecular marker to assess cytokinesis. METHODOLOGY: The morphological events and their timing during the cell cycle of T. rangeli epimastigotes were assessed using DNA staining, flagellum labelling and bromodeoxyuridine incorporation. Messenger RNA levels of four genes previously associated with the cell cycle of trypanosomatids (AUK1, PLK, MOB1 and TRACK) were evaluated in the different T. rangeli forms. FINDINGS: T. rangeli epimastigotes completed the cell cycle in vitro in 20.8 h. PLK emerged as a potential molecular marker for cell division, as its mRNA levels were significantly increased in exponentially growing epimastigotes compared with growth-arrested parasites or in vitro-differentiated trypomastigotes. PLK expression in T. rangeli can be detected near the flagellum protrusion site, reinforcing its role in the cell cycle. Interestingly, T. rangeli bloodstream trypomastigotes exhibited very low mRNA levels of PLK and were almost entirely composed of parasites in G1 phase. MAIN CONCLUSIONS: Our work is the first to describe the T. rangeli cell cycle in vitro and proposes that PLK mRNA levels could be a useful tool to investigate the T. rangeli ability to proliferate within the mammalian host bloodstream.

Ribotypes associated with Clostridium difficile outbreaks in Brazil display distinct surface protein profiles.

Clostridium difficile is a spore-forming anaerobic intestinal pathogen that causes Clostridium difficile infection (CDI). C. difficile is the leading cause of toxin-mediated nosocomial antibiotic-associated diarrhea. The pathogenesis of CDI is attributed to two major virulence factors, TcdA and TcdB toxins, that cause the symptomatic infection. C. difficile also expresses a number of key proteins, including cell wall proteins (CWPs). S-layer proteins (SLPs) are CWPs that form a paracrystalline surface array that coats the surface of the bacterium. SLPs have a role in C. difficile binding to the gastrointestinal tract, but their importance in virulence need to be better elucidated. Here, we describe bottom-up proteomics analysis of surface-enriched proteins fractions obtained through glycine extraction of five C. difficile clinical isolates from Brazil using gel-based and gel-free approaches. We were able to identify approximately 250 proteins for each strain, among them SlpA, Cwp2, Cwp6, CwpV and Cwp84. Identified CWPs presented different amino acid coverage, which might suggest differences in post-translational modifications. Proteomic analysis of SLPs from ribotype 133, agent of C. difficile outbreaks in Brazil, revealed unique proteins and provided additional information towards in depth characterization of the strains causing CDI in Brazil.

Detection of biomarkers of pathogenic Naegleria fowleri through mass spectrometry and proteomics.

Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix-assisted laser-desorption-ionization-time-of-flight mass spectrometry MALDI-TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF-TOF instrument. MALDI-TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI-TOF MS fingerprinting is a rapid, reproducible, high-throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents.

Shotgun proteomics of detergent-solubilized proteins from Trypanosoma evansi.

Trypanosoma evansi, the causative agent of surra, is the most prevalent pathogenic salivarian trypanosome and affects the majority of domesticated and wild animals in endemic regions. This work aimed to analyze detergent-solubilized T. evansi proteins and identify potential diagnostic biomarkers for surra. Triton X-114-extracted membrane-enriched proteins (MEP) of T. evansi bloodstream forms were analyzed using a gel-free technique (LC-ESI-MS/MS). 247 proteins were identified following the MS analysis of three biological and technical replicates. Two of these proteins were predicted to have a GPI-anchor, 100 (40%) were predicted to have transmembrane domains, and 166 (67%) were predicted to be membrane-bound based on at least one of six features: location (WolfPSORT, DeepLoc-2.0, Protcomp-9.0), transmembrane, GPI, and gene ontology. It was predicted that 76 (30%) of proteins had membrane evidence. Typical membrane proteins for each organelle were identified, among them ISG families (64, 65, and 75 kDa), flagellar calcium-binding protein, 24 kDa calflagin, syntaxins and oligosaccharyltransferase some of which had previously been studied in other trypanosomatids. T. evansi lacks singletons and exclusive orthologous groups, whereas three distinct epitopes have been identified. Data are available via ProteomeXchange with identifier PXD040594. SIGNIFICANCE: Trypanosoma evansi is a highly prevalent parasite that induces a pathological condition known as "surra" in various species of ungulates across five continents. The infection gives rise to symptoms that are not pathognomonic, thereby posing challenges in its diagnosis and leading to substantial economic losses in the livestock industry. A significant challenge arises from the absence of a diagnostic test capable of distinguishing between Trypanosoma equiperdum and T. evansi, both of which are implicated in equine diseases. Therefore, there is a pressing need to conduct research on the biochemistry of the parasite in order to identify proteins that could potentially serve as targets for differential diagnosis or therapeutic interventions.

Quantification of immunoreactive viral influenza proteins by immunoaffinity capture and isotope-dilution liquid chromatography-tandem mass spectrometry.

An immunocapture isotope dilution mass spectrometry (IC-IDMS) method was developed to quantify antibody-bound influenza hemagglutinins (HA) in trivalent influenza vaccines (TIV). Currently, regulatory potency requirements for TIV require HA quantification based on the single radial immunodiffusion (SRID) assay, which is time-consuming, laborious, and requires production of large quantities of reagents globally. In IC-IDMS, antiserum to the HA of interest captured viral proteins that were in the correct conformation to be recognized by the antibodies. The captured proteins were digested, and evolutionarily conserved tryptic peptides were quantified using isotope-dilution liquid chromatography-tandem mass spectrometry. IC-IDMS relies on antibody-antigen binding similar to SRID but incorporates the accuracy and precision of IDMS. Polyclonal antibodies (pAb-H3) prepared by injection of sheep with purified H3 HA captured 82.9% (55.26 fmol/µL) of the total H3 HA (66.69 fmol/µL) from the commercial TIV and 93.6% (57.23 fmol/µL) of the total H3 HA (61.14 fmol/µL) in purified virus. While other HA (H1, B), neuraminidase (N1, N2, NB), viral matrix proteins, and nucleoproteins were also captured by this antiserum, our results were not affected due to the specificity of the mass spectrometer. IC-IDMS is an accurate, precise, sensitive, and selective method to measure antibody-bound HA in purified virus and commercial vaccines.

A historical and proteomic analysis of botulinum neurotoxin type/G.

BACKGROUND: Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex. RESULTS: An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs--nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison. CONCLUSIONS: The in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MS(E) data analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.

Quantitative mass spectrometry for bacterial protein toxins--a sensitive, specific, high-throughput tool for detection and diagnosis.

Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.

Exoproteomic analysis of two MLST clade 2 strains of Clostridioides difficile from Latin America reveal close similarities.

Clostridioides difficile BI/NAP1/ribotype 027 is an epidemic hypervirulent strain found worldwide, including in Latin America. We examined the genomes and exoproteomes of two multilocus sequence type (MLST) clade 2 C. difficile strains considered hypervirulent: ICC-45 (ribotype SLO231/UK[CE]821), isolated in Brazil, and NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica. C. difficile isolates were cultured and extracellular proteins were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Genomic analysis revealed that these isolates shared most of the gene composition. Only 83 and 290 NAP1/027 genes were considered singletons in ICC-45 and NAP1/027, respectively. Exoproteome analysis revealed 197 proteins, of which 192 were similar in both strains. Only five proteins were exclusive to the ICC-45 strain. These proteins were involved with catalytic and binding functions and indirectly interacted with proteins related to pathogenicity. Most proteins, including TcdA, TcdB, flagellin subunit, and cell surface protein, were overrepresented in the ICC-45 strain; 14 proteins, including mature S-layer protein, were present in higher proportions in LIBA5756. Data are available via ProteomeXchange with identifier PXD026218. These data show close similarity between the genome and proteins in the supernatant of two strains with hypervirulent features isolated in Latin America and underscore the importance of epidemiological surveillance of the transmission and emergence of new strains.

Mass spectrometric analysis of multiple pertussis toxins and toxoids.

Bordetella pertussis (Bp) is the causative agent of pertussis, a vaccine preventable disease occurring primarily in children. In recent years, there has been increased reporting of pertussis. Current pertussis vaccines are acellular and consist of Bp proteins including the major virulence factor pertussis toxin (Ptx), a 5-subunit exotoxin. Variation in Ptx subunit amino acid (AA) sequence could possibly affect the immune response. A blind comparative mass spectrometric (MS) analysis of commercially available Ptx as well as the chemically modified toxoid (Ptxd) from licensed vaccines was performed to assess peptide sequence and AA coverage variability as well as relative amounts of Ptx subunits. Qualitatively, there are similarities among the various sources based on AA percent coverages and MS/MS fragmentation profiles. Additionally, based on a label-free mass spectrometry-based quantification method there is differential relative abundance of the subunits among the sources.

Taenia crassiceps cysticerci: Characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci.

Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14 gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101- to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14 gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14 gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T. solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14 gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T. solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays.

Dynamics of protein synthesis in the initial steps of strobilation in the model cestode parasite Mesocestoides corti (syn. vogae).

Mesocestoides corti (syn. vogae) is a useful model for developmental studies of platyhelminth parasites of the Cestoda class, such as Taenia spp. or Echinococcus spp. It has been used in studies to characterize cestode strobilation, i.e. the development of larvae into adult worms. So far, little is known about the initial molecular events involved in cestode strobilation and, therefore, we carried out a study to characterize newly synthesized (NS) proteins upon strobilation induction. An approach based on bioorthogonal noncanonical amino acid tagging and mass spectrometry was used to label, isolate, identify, and quantify NS proteins in the initial steps of M. corti strobilation. Overall, 121 NS proteins were detected exclusively after induction of strobilation, including proteins related to development pathways, such as insulin and notch signaling. Metabolic changes that take place in the transition from the larval stage to adult worm were noted in special NS protein subsets related to developmental processes, such as focal adhesion, cell leading edge, and maintenance of location. The data shed light on mechanisms underlying early steps of cestode strobilation and enabled identification of possible developmental markers. We also consider the use of developmental responsive proteins as potential drug targets for developing novel anthelmintics. BIOLOGICAL SIGNIFICANCE: Larval cestodiases are life-threatening parasitic diseases that affect both man and domestic animals worldwide. Cestode parasites present complex life cycles, in which they undergo major morphological and physiological changes in the transition from one life-stage to the next. One of these transitions occurs during cestode strobilation, when the mostly undifferentiated and non-segmented larval or pre-adult form develops into a fully segmented and sexually differentiated (strobilated) adult worm. Although the proteomes of bona fide larvae and strobialted adults have been previously characterized for a few cestode species, little is still known about the dynamic of protein synthesis during the early steps of cestode strobilation. Now, the assessment of newly synthesized (NS) proteins within the first 48 h of strobilation the model cestode M. corti allowed to shed light on molecular mechanisms that are triggered by strobilation induction. The functional analyses of this repertoire of over a hundred NS proteins pointed out to changes in metabolism and activation of classical developmental signaling pathways in early strobilation. Many of the identified NS proteins may become valuable cestode developmental markers and their involvement in vital processes make them also good candidate targets for novel anthelmintic drugs.

Differential responses to stress of two Mycoplasma hyopneumoniae strains.

Mycoplasma hyopneumoniae is a respiratory pathogen, causing porcine enzootic pneumonia. To survive in the porcine respiratory tract, M. hyopneumoniae must cope with both oxidative and heat stress imposed by the host. To get insights into M. hyopneumoniae stress responses and pathogenicity mechanisms, the protein profiles of two M. hyopneumoniae strains, pathogenic 7448 strain and non-pathogenic strain J, were surveyed under oxidative (OS) or heat (HS) stress. M. hyopneumoniae strains were submitted to OS (0.5% hydrogen peroxide) or HS (temperature shifts to 42 °C) conditions and protein profiling was carried out by LC-MS/MS and label-free quantitative analyses. Data are available via ProteomeXchange with identifier PXD012742. Qualitative and quantitative differences involving 40-60 M. hyopneumoniae proteins were observed for both strains when comparing bacteria exposed to OS or HS to non-treated controls. However, no differences in abundance were found in proteins classically related to stress responses, as peroxidases and chaperones, suggesting that these proteins would be constitutively present in both strains in the tested conditions. Interestingly, under stress conditions, more virulence-related proteins were detected in M. hyopneumoniae 7448 differentially represented proteins than in M. hyopneumoniae J, suggesting that stress may trigger a differential response of the corresponding genes, shared by both strains.

Unraveling oxidative stress response in the cestode parasite Echinococcus granulosus.

Cystic hydatid disease (CHD) is a worldwide neglected zoonotic disease caused by Echinococcus granulosus. The parasite is well adapted to its host by producing protective molecules that modulate host immune response. An unexplored issue associated with the parasite's persistence in its host is how the organism can survive the oxidative stress resulting from parasite endogenous metabolism and host defenses. Here, we used hydrogen peroxide (H2O2) to induce oxidative stress in E. granulosus protoescoleces (PSCs) to identify molecular pathways and antioxidant responses during H2O2 exposure. Using proteomics, we identified 550 unique proteins; including 474 in H2O2-exposed PSCs (H-PSCs) samples and 515 in non-exposed PSCs (C-PSCs) samples. Larger amounts of antioxidant proteins, including GSTs and novel carbonyl detoxifying enzymes, such as aldo-keto reductase and carbonyl reductase, were detected after H2O2 exposure. Increased concentrations of caspase-3 and cathepsin-D proteases and components of the 26S proteasome were also detected in H-PSCs. Reduction of lamin-B and other caspase-substrate, such as filamin, in H-PSCs suggested that molecular events related to early apoptosis were also induced. We present data that describe proteins expressed in response to oxidative stress in a metazoan parasite, including novel antioxidant enzymes and targets with potential application to treatment and prevention of CHD.

Differential secretome profiling of a swine tracheal cell line infected with mycoplasmas of the swine respiratory tract.

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar. However, M. hyopneumoniae causes porcine enzootic pneumonia, while M. flocculare is a commensal bacterium. M. hyopneumoniae and M. flocculare do not penetrate their host cells, and secreted proteins are important for bacterium-host interplay. Thus, the secretomes of a swine trachea cell line (NPTr) infected with M. hyopneumoniae 7448 (a pathogenic strain), M. hyopneumoniae J (a non-pathogenic strain) and M. flocculare were compared to shed light in bacterium-host interactions. Medium from the cultures was collected, and secreted proteins were identified by a LC-MS/MS. Overall numbers of identified host and bacterial proteins were, respectively, 488 and 58, for NPTr/M. hyopneumoniae 7448; 371 and 67, for NPTr/M. hyopneumoniae J; and 203 and 81, for NPTr/M. flocculare. The swine cells revealed different secretion profiles in response to the infection with each M. hyopneumoniae strain or with M. flocculare. DAMPs and extracellular proteasome proteins, secreted in response to cell injury and death, were secreted by NPTr cells infected with M. hyopneumoniae 7448. All three mycoplasmas secreted virulence factors during NPTr infection, but M. hyopneumoniae 7448 secreted higher number of adhesins and hypothetical proteins, that may be related with pathogenicity. SIGNIFICANCE: The enzootic pneumonia caused by mycoplasmas of swine respiratory tract has economic loss consequences in pig industry due to antibiotic costs and pig weight loss. However, some genetically similar mycoplasmas are pathogenic while others, such as Mycoplasma hyopneumoniae and Mycoplasma flocculare, are non-pathogenic. Here, we conducted an infection assay between swine cells and pathogenic and non-pathogenic mycoplasmas to decipher secreted proteins during host-pathogen interaction. Mycoplasma response to cell infection was also observed. Our study provided new insights on secretion profile of swine cells in response to the infection with pathogenic and non-pathogenic mycoplasmas. It was possible to observe that pathogenic M. hyopneumoniae 7448 secreted known virulence factors and swine cells responded by inducing cell death. Otherwise, M. hyopneumoniae J and M. flocculare, non-pathogenic mycoplasmas, secreted a different profile of virulence factors in response to swine cells. Consequently, swine cells altered their secretome profile, but the changes were not sufficient to cause disease.

Lessons from the reestablishment of Public Health Laboratory activities in Puerto Rico after Hurricane Maria.

Public Health Laboratories (PHLs) in Puerto Rico did not escape the devastation caused by Hurricane Maria. We implemented a quality management system (QMS) approach to systematically reestablish laboratory testing, after evaluating structural and functional damage. PHLs were inoperable immediately after the storm. Our QMS-based approach began in October 2017, ended in May 2018, and resulted in the reestablishment of 92% of baseline laboratory testing capacity. Here, we share lessons learned from the historic recovery of the largest United States' jurisdiction to lose its PHL capacity, and provide broadly applicable tools for other jurisdictions to enhance preparedness for public health emergencies.