Comparative Exoproteome Analysis of Streptococcus suis Human Isolates

The swine pathogen Streptococcus suis is a Gram-positive bacterium which causes infections in pigs, with an impact in animal health and in the livestock industry, and it is also an important zoonotic agent. During the infection process, surface and secreted proteins are essential in the interaction between microorganisms and their hosts. Here, we report a comparative proteomic analysis of the proteins released to the extracellular milieu in six human clinical isolates belonging to the highly prevalent and virulent serotype 2. The total secreted content was precipitated and analyzed by GeLC-MS/MS. In the six strains, 144 proteins assigned to each of the categories of extracellular or surface proteins were identified, as well as 680 predicted cytoplasmic proteins, many of which are putative moonlighting proteins. Of the nine predicted signal peptide-I secreted proteins, seven had relevant antigenic potential when they were analyzed through bioinformatic analysis. This is the first work comparing the exoproteome fraction of several human isolates of this important pathogen.

Genomic insights on DNase production in Streptococcus agalactiae ST17 and ST19 strains

Streptococcus agalactiae evasion from the human defense mechanisms has been linked to the production of DNases. These were proposed to contribute to the hypervirulence of S. agalactiae ST17/capsular-type III strains, mostly associated with neonatal meningitis. We performed a comparative genomic analysis between ST17 and ST19 human strains with different cell tropism and distinct DNase production phenotypes. All S. agalactiae ST17 strains, with the exception of 2211-04, were found to display DNase activity, while the opposite scenario was observed for ST19, where 1203-05 was the only DNase(+) strain. The analysis of the genetic variability of the seven genes putatively encoding secreted DNases in S. agalactiae revealed an exclusive amino acid change in the predicted signal peptide of GBS0661 (NucA) of the ST17 DNase(-), and an exclusive amino acid change alteration in GBS0609 of the ST19 DNase(+) strain. Further core-genome analysis identified some specificities (SNVs or indels) differentiating the DNase(-) ST17 2211-04 and the DNase(+) ST19 1203-05 from the remaining strains of each ST. The pan-genomic analysis evidenced an intact phage without homology in S. agalactiae and a transposon homologous to TnGBS2.3 in ST17 DNase(-) 2211-04; the transposon was also found in one ST17 DNase(+) strain, yet with a different site of insertion. A group of nine accessory genes were identified among all ST17 DNase(+) strains, including the Eco47II family restriction endonuclease and the C-5 cytosine-specific DNA methylase. None of these loci was found in any DNase(-) strain, which may suggest that these proteins might contribute to the lack of DNase activity. In summary, we provide novel insights on the genetic diversity between DNase(+) and DNase(-) strains, and identified genetic traits, namely specific mutations affecting predicted DNases (NucA and GBS0609) and differences in the accessory genome, that need further investigation as they may justify distinct DNase-related virulence phenotypes in S. agalactiae.

Serum Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) Level as a Potential Biomarker of Cholangiocarcinoma

Diagnostic and/or prognostic biomarkers for cholangiocarcinoma (CCA) are still insufficient with poor prognosis of patients. To discover a new CCA biomarker, we constructed our secretome database of three CCA cell lines and one control cholangiocyte cell line using GeLC-MS/MS. We selected candidate proteins by five bioinformatics tools for secretome analysis. The inclusion criteria were as follows: having predicted signal peptide or being predicted as non-classically secreted protein; together with having no transmembrane helix and being previously detected in plasma and having the highest number of signal peptide cleavage sites. Eventually, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) was selected for further analysis. To validate APEX1 as a bio-marker for CCA, serum APEX1 levels of 80, 39, and 40 samples collected from CCA, benign biliary diseases (BBD), and healthy control groups, respectively, were measured using dot blot analysis. The results showed that serum APEX1 level in CCA group was significantly higher than that in BBD or healthy control group. Among CCA patients, serum APEX1 level was significantly higher in patients having metastasis than in those without metastasis. The higher level of serum APEX1 was correlated with the shorter survival time of the patients. Serum APEX1 level might be a diagnostic and prognostic biomarker for CCA.

The Functional Characterization of Podosphaera xanthii Candidate Effector Genes Reveals Novel Target Functions for Fungal Pathogenicity

Podosphaera xanthii is the main causal agent of powdery mildew disease in cucurbits. In a previous study, we determined that P. xanthii expresses approximately 50 Podosphaera effector candidates (PECs), identified based on the presence of a predicted signal peptide and the absence of functional annotation. In this work, we used host-induced gene silencing (HIGS), employing Agrobacterium tumefaciens as a vector for the delivery of the silencing constructs (ATM-HIGS), to identify genes involved in early plant-pathogen interaction. The analysis of seven selected PEC-encoding genes showed that six of them, PEC007, PEC009, PEC019, PEC032, PEC034, and PEC054, are required for P. xanthii pathogenesis, as revealed by reduced fungal growth and increased production of hydrogen peroxide by host cells. In addition, protein models and protein-ligand predictions allowed us to identify putative functions for these candidates. The biochemical activities of PEC019, PEC032, and PEC054 were elucidated using their corresponding proteins expressed in Escherichia coli. These proteins were confirmed as phospholipid-binding protein, α-mannosidase, and cellulose-binding protein. Further, BLAST searches showed that these three effectors are widely distributed in phytopathogenic fungi. These results suggest novel targets for fungal effectors, such as host-cell plasma membrane, host-cell glycosylation, and damage-associated molecular pattern–triggered immunity.

The hydrophobin-like OmSSP1 may be an effector in the ericoid mycorrhizal symbiosis

Mutualistic and pathogenic plant-colonizing fungi use effector molecules to manipulate the host cell metabolism to allow plant tissue invasion. Some small secreted proteins (SSPs) have been identified as fungal effectors in both ectomycorrhizal and arbuscular mycorrhizal fungi, but it is currently unknown whether SSPs also play a role as effectors in other mycorrhizal associations. Ericoid mycorrhiza is a specific endomycorrhizal type that involves symbiotic fungi mostly belonging to the Leotiomycetes (Ascomycetes) and plants in the family Ericaceae. Genomic and RNASeq data from the ericoid mycorrhizal fungus Oidiodendron maius led to the identification of several symbiosis-upregulated genes encoding putative SSPs. OmSSP1, the most highly symbiosis up-regulated SSP, was found to share some features with fungal hydrophobins, even though it lacks the Pfam hydrophobin domain. Sequence alignment with other hydrophobins and hydrophobin-like fungal proteins placed OmSSP1 within Class I hydrophobins. However, the predicted features of OmSSP1 may suggest a distinct type of hydrophobin-like proteins. The presence of a predicted signal peptide and a yeast-based signal sequence trap assay demonstrate that OmSSP1 is secreted during symbiosis. OmSSP1 null-mutants showed a reduced capacity to form ericoid mycorrhiza with Vaccinium myrtillus roots, suggesting a role as effectors in the ericoid mycorrhizal interaction.

Only a small subset of the SPRY domain gene family in Globodera pallida is likely to encode effectors, two of which suppress host defences induced by the potato resistance gene Gpa2

Analysis of the genome sequence of the potato cyst nematode, Globodera pallida, has shown that a substantial gene family (approximately 300 sequences) of proteins containing a SPRY domain is present in this species. This is a huge expansion of the gene family as compared to other organisms, including other plant-parasitic nematodes. Some SPRY domain proteins from G. pallida and G. rostochiensis have signal peptides for secretion and are deployed as effectors. One of these SPRYSEC proteins has been shown to suppress host defence responses. We describe further analysis of this gene family in G. pallida. We show that only a minority (10%) of the SPRY domain proteins in this species have a predicted signal peptide for secretion and that the presence of a signal peptide is strongly correlated with the corresponding gene being expressed at the early stages of parasitism. The data suggest that while the gene family is greatly expanded, only a minority of SPRY domain proteins in G. pallida are SPRYSEC candidate effectors. We show that several new SPRYSECs from G. pallida are expressed in the dorsal gland cell and demonstrate that some, but not all, of the SPRYSECs can suppress the hypersensitive response induced by co-expression of the resistance gene Gpa2 and its cognate avirulence factor RBP-1 in Nicotiana benthamiana.

A Novel Hormone Is Required for the Development of Reproductive Phenotypes in Adult Female Crabs

The crustacean male-specific androgenic hormone is widely accepted as a key factor in sexual differentiation and in the development of secondary sex characteristics. However, the mechanism by which the plethora of different reproductive strategies are controlled and executed in crustaceans is not known. We discovered in the blue crab, Callinectes sapidus, a hitherto unknown neurohormone, named crustacean female sex hormone (CFSH), in distinct neurosecretory cells in the eyestalk ganglia. CFSH is highly expressed in females but weakly in males, and its crucial role in developing adult female phenotypes has now been established. CFSH cDNA encodes a 225-amino acid (aa) novel protein composed of a 23-aa predicted signal peptide, 33-aa precursor-related peptide and 167-aa mature protein that did not match any other sequence in GenBank. CFSH RNA interference knockdown by multiple administrations of double-stranded RNA at the prepubertal stage causes abnormal development of brooding and mating systems upon puberty. These systems include a pair of gonopores and an egg attachment system for brooding, comprised of an enlarged semicircular abdomen and ovigerous setae. The ovigerous setae in CFSH knocked-down females were fewer and 50% shorter and the gonopores were either significantly smaller than those of controls, misplaced, or absent. We also identified CFSH in the green crab, Carcinus maenas, a species that shares a similar reproductive strategy with C. sapidus. Together, our data provide the first evidence for the presence of a female hormone in crustaceans and its importance in positively controlling anatomic features associated with brooding and mating systems. From an evolutionary standpoint, the endocrine control supporting a female-specific reproductive strategy, as previously described for many vertebrate species, has now been demonstrated for the first time in crustaceans.

Bioinformatics Analysis and Characteristics of UL17 Protein Encoded by the UL17 Gene from Duck Enteritis Virus

Previous studies indicate that the UL17 protein and its homology play similar roles in viral DNA cleavage and packaging of herpesviruses. However, there is no report on the UL17 gene product of DEV. Here in this article we used various bioinformatics softwares and online web servers to analyze the basic properties of the DEV UL17 protein. The results showed that the DEV UL17 protein was 75.94298 kDa, consisting of 684 amino acids. It had multiple motif sites, phosphorylation sites and antigenic epitopes. There were no predicted signal peptide sites or transmembrane regions in DEV UL17 protein. The results of secondary structure and teriary structure were exhibited in the pictures in this article. These properties of the DEV UL17 protein provide a prerequisite for further functional analysis of this gene.

Suppression of Bamboo Mosaic Virus Accumulation by a Putative Methyltransferase in Nicotiana benthamiana

ABSTRACT Bamboo mosaic virus (BaMV) is a 6.4-kb positive-sense RNA virus belonging to the genus Potexvirus of the family Flexiviridae. The 155-kDa viral replicase, the product of ORF1, comprises an N-terminal S-adenosyl-l-methionine (AdoMet)-dependent guanylyltransferase, a nucleoside triphosphatase/RNA 5′-triphosphatase, and a C-terminal RNA-dependent RNA polymerase (RdRp). To search for cellular factors potentially involved in the regulation of replication and/or transcription of BaMV, the viral RdRp domain was targeted as bait to screen against a leaf cDNA library of Nicotiana benthamiana using a yeast two-hybrid system. A putative methyltransferase (PNbMTS1) of 617 amino acid residues without an established physiological function was identified. Cotransfection of N. benthamiana protoplasts with a BaMV infectious clone and the PNbMTS1-expressing plasmid showed a PNbMTS1 dosage-dependent inhibitory effect on the accumulation of BaMV coat protein. Deletion of the N-terminal 36 amino acids, deletion of a predicted signal peptide or transmembrane segment, or mutations in the putative AdoMet-binding motifs of PNbMTS1 abolished the inhibitory effect. In contrast, suppression of PNbMTS1 by virus-induced gene silencing in N. benthamiana increased accumulation of the viral coat protein as well as the viral genomic RNA. Collectively, PNbMTS1 may function as an innate defense protein against the accumulation of BaMV through an uncharacterized mechanism.

Urease Produced by Coccidioides posadasii Contributes to the Virulence of This Respiratory Pathogen

ABSTRACT Urease activity during in vitro growth in the saprobic and parasitic phases of Coccidioides spp. is partly responsible for production of intracellular ammonia released into the culture media and contributes to alkalinity of the external microenvironment. Although the amino acid sequence of the urease of Coccidioides posadasii lacks a predicted signal peptide, the protein is transported from the cytosol into vesicles and the central vacuole of parasitic cells (spherules). Enzymatically active urease is released from the contents of mature spherules during the parasitic cycle endosporulation stage. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense host inflammatory response. Ammonia production by the spherules of C. posadasii is markedly increased by the availability of exogenous urea found in relatively high concentrations at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of C. posadasii resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. BALB/c mice challenged intranasally with the mutant strain showed increased survival, a well-organized granulomatous response to infection, and better clearance of the pathogen than animals challenged with either the parental or the reconstituted (revertant) strain. We conclude that ammonia and enzymatically active urease released from spherules during the parasitic cycle of C. posadasii contribute to host tissue damage, which exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen.