Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides

<p>Abstract</p> <p>Background</p> <p>Despite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach.</p> <p>Results</p> <p>The cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipμ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses.</p> <p>Conclusions</p> <p>The proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis.</p

Investigation of the potential of Brevibacillus spp. for the biosynthesis of nonribosomally produced bioactive compounds by combination of genome mining with MALDI-TOF mass spectrometry

The biosynthetic potential of 11 Brevibacillus spp. strains was investigated by combination of genome mining with mass spectrometric analysis using MALDI-TOF mass spectrometry. These endophytic, plant associated Brevibacillus strains were isolated from crop plants, such as coffee and black pepper, in Vietnam. Draft genomes of these strains were available. They were classified (a) by comparison with type strains and a collection of genome-sequenced Brevibacillus spp. deposited in the NCBI data base as well as (b) by construction of a phylogenetic tree from the core sequences of publicly available genomes of Brevibacillus strains. They were identified as Brevibacillus brevis (1 strain); parabrevis (2 strains); porteri (3 strains); and 5 novel Brevibacillus genomospecies. Our work was specifically focused on the detection and characterization of nonribosomal peptides produced by these strains. Structural characterization of these compounds was performed by LIFT-MALDI-TOF/TOF mass spectrometric sequence analysis. The highlights of our work were the demonstration of the tyrocidines, a well-known family of cyclodecapeptides of great structural variability, as the main products of all investigated strains and the identification of a novel class of pentapeptides produced by B. brevis; B. schisleri; and B. porteri which we designate as brevipentins. Our biosynthetic studies demonstrate that knowledge of their biosynthetic capacity can efficiently assist classification of Brevibacillus species.Peer Reviewe

Perchlorate-specific proteomic stress responses of Debaryomyces hansenii could enable microbial survival in Martian brines

If life exists on Mars, it would face several challenges including the presence of perchlorates, which destabilize biomacromolecules by inducing chaotropic stress. However, little is known about perchlorate toxicity for microorganisms on the cellular level. Here, we present the first proteomic investigation on the perchlorate-specific stress responses of the halotolerant yeast Debaryomyces hansenii and compare these to generally known salt stress adaptations. We found that the responses to NaCl and NaClO4-induced stresses share many common metabolic features, for example, signalling pathways, elevated energy metabolism, or osmolyte biosynthesis. Nevertheless, several new perchlorate-specific stress responses could be identified, such as protein glycosylation and cell wall remodulations, presumably in order to stabilize protein structures and the cell envelope. These stress responses would also be relevant for putative life on Mars, which—given the environmental conditions—likely developed chaotropic defence strategies such as stabilized confirmations of biomacromolecules or the formation of cell clusters.Peer Reviewe

Silence as a way of niche adaptation: mecC-MRSA with variations in the accessory gene regulator (agr) functionality express kaleidoscopic phenotypes

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I–IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches.Peer Reviewe

The role of facemasks and hand hygiene in the prevention of influenza transmission in households: results from a cluster randomised trial; Berlin, Germany, 2009-2011

Background: Previous controlled studies on the effect of non-pharmaceutical interventions (NPI) - namely the use of facemasks and intensified hand hygiene - in preventing household transmission of influenza have not produced definitive results. We aimed to investigate efficacy, acceptability, and tolerability of NPI in households with influenza index patients. Methods: We conducted a cluster randomized controlled trial during the pandemic season 2009/10 and the ensuing influenza season 2010/11. We included households with an influenza positive index case in the absence of further respiratory illness within the preceding 14 days. Study arms were wearing a facemask and practicing intensified hand hygiene (MH group), wearing facemasks only (M group) and none of the two (control group). Main outcome measure was laboratory confirmed influenza infection in a household contact. We used daily questionnaires to examine adherence and tolerability of the interventions. Results: We recruited 84 households (30 control, 26 M and 28 MH households) with 82, 69 and 67 household contacts, respectively. In 2009/10 all 41 index cases had a influenza A (H1N1) pdm09 infection, in 2010/11 24 had an A (H1N1) pdm09 and 20 had a B infection. The total secondary attack rate was 16% (35/218). In intention-totreat analysis there was no statistically significant effect of the M and MH interventions on secondary infections. When analysing only households where intervention was implemented within 36 h after symptom onset of the index case, secondary infection in the pooled M and MH groups was significantly lower compared to the control group (adjusted odds ratio 0.16, 95% CI, 0.03-0.92). In a per-protocol analysis odds ratios were significantly reduced among participants of the M group (adjusted odds ratio, 0.30, 95% CI, 0.10-0.94). With the exception of MH index cases in 2010/11 adherence was good for adults and children, contacts and index cases. Conclusions: Results suggest that household transmission of influenza can be reduced by the use of NPI, such as facemasks and intensified hand hygiene, when implemented early and used diligently. Concerns about acceptability and tolerability of the interventions should not be a reason against their recommendation

Perspective on Proteomics for Virus Detection in Clinical Samples

One of the most widely used methods to detect an acute viral infection in clinical specimens is diagnostic real-time polymerase chain reaction. However, because of the COVID-19 pandemic, mass-spectrometry-based proteomics is currently being discussed as a potential diagnostic method for viral infections. Because proteomics is not yet applied in routine virus diagnostics, here we discuss its potential to detect viral infections. Apart from theoretical considerations, the current status and technical limitations are considered. Finally, the challenges that have to be overcome to establish proteomics in routine virus diagnostics are highlighted.Peer Reviewe

Recombinant AcnB, NrdR and RibD of Acinetobacter baumannii and their potential interaction with DNA adenine methyltransferase AamA

In the last decades Acinetobacter baumannii developed into an increasingly challenging nosocomial pathogen.A. baumannii ATCC 17978 harbors a DNA-(adenine N6)-methyltransferase termed AamA. Previous studiesrevealed a low specific activity of AamA in vitro despite proven folding, which led us to speculate about possibleinteraction partners assisting AamA in targeting methylation sites. Here, applying a pulldown assay with sub-sequent mass spectrometry we identified aconitate hydratase 2 (AcnB) as possible interaction partner. In addi-tion, we considered the putative transcriptional regulator gene nrdR (A1S_0220) and the pyrimidine deaminase/reductase gene ribD (A1S_0221) of A. baumannii strain ATCC 17978 to encode additional potential interactionpartners due to their vicinity to the aamA gene (A1S_0222). Proteins were recombinantly produced in themilligram scale, purified to near homogeneity, and interactions with AamA were studied applying blue native gelelectrophoreses, electrophoretic mobility shift assay, chemical cross-linking and co-immunoprecipitation. Theseanalyses did not provide evidence of interaction between AamA and purified proteins. Solution structures ofRibD, NrdR and AcnB were studied by small-angle X-ray scattering (SAXS) alone and in combination with AamA.While in the case of RibD and AcnB no evidence of an interaction with AamA was produced, addition of AamA toNrdR resulted in dissociation of long and rod-shaped polymeric NrdR structures, implying a specific but transientinteraction. Moreover, we identified a molecular crowding effect possibly impeding the DNA methyltransferaseactivity in vivo and a sequence-independent DNA binding activity of AamA calling for continued efforts toidentify the interaction network of AamA

Global ubiquitination analysis reveals extensive modification and proteasomal degradation of cowpox virus proteins, but preservation of viral cores

The emergence of Variola virus-like viruses by natural evolution of zoonotic Orthopoxviruses, like Cowpox virus (CPXV), is a global health threat. The proteasome is essential for poxvirus replication, making the viral components interacting with the ubiquitin-proteasome system attractive antiviral targets. We show that proteasome inhibition impairs CPXV replication by prevention of uncoating, suggesting that uncoating is mediated by proteasomal degradation of viral core proteins. Although Orthopoxvirus particles contain considerable amounts of ubiquitin, distinct modification sites are largely unknown. Therefore, for the first time, we analyzed globally ubiquitination sites in CPXV mature virion proteins using LC-MS/MS. Identification of 137 conserved sites in 54 viral proteins among five CPXV strains revealed extensive ubiquitination of structural core proteins. Moreover, since virions contained primarily K48-linked polyubiquitin, we hypothesized that core proteins are modified accordingly. However, quantitative analysis of ubiquitinated CPXV proteins early in infection showed no proteasomal degradation of core proteins. Instead, our data indicate that the recently suggested proteasomal regulation of the uncoating factor E5 is a prerequisite for uncoating. Expanding our understanding of poxvirus uncoating and elucidating a multitude of novel ubiquitination sites in poxvirus proteins, the present study verifies the major biological significance of ubiquitin in poxvirus infection

Analysis of putative OPV mature virion-associated cellular proteins.

<p>The distribution of the protein copy numbers of viral and human proteins is shown in A. In order to discriminate between putative MV-associated human proteins and contaminants, the protein abundances within the MV preparations and the abundances in HepG2 cells were used for 1D and 2D annotation enrichment analysis. For the 2D analysis, normalized iBAQ values of the OPV MV and HepG2 cells were used. The score distribution of enriched GO terms with a FDR below 0.01 is plotted in C. Terms along the diagonal (black) are enriched or depleted in the MV and the cells in the same way and represent cellular background. The GO terms on the right side of the diagonal are enriched in the MV relative to the cell, and therefore represent putative MV-associated human proteins. If the proteins behind the GO terms are associated with increasing abundance in the OPV MV the terms are labelled blue, if they are associated with increasing abundance in the MV and associated with low abundance in the cells the terms are labelled red and if they are associated with low abundance in the cells the terms are labelled green. For 1D annotation enrichment analysis, iBAQ ratios for the human proteins in the MV preparations relative to the abundance in HepG2 cells were calculated (B). The score distribution of GO terms enriched in the MV preparations with a FDR below 0.01 is shown in D, redundant terms were removed from the Fig but can be found in Table L in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141527#pone.0141527.s003" target="_blank">S1 File</a>.</p