Vishnu M. Dhople

Time-resolved quantitative proteome profiling of host–pathogen interactions: The response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells

Staphylococcus aureus is a versatile Gram‐positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse‐chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on‐membrane digestion, and high‐sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof‐of‐principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.

Tyrosine phosphoproteome of hamster spermatozoa : Role of glycerol-3-phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine‐phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol‐3‐phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation.

Comparative evaluation of saliva collection methods for proteome analysis.

BACKGROUND Saliva collection devices are widely used for large-scale screening approaches. This study was designed to compare the suitability of three different whole-saliva collection approaches for subsequent proteome analyses. METHODS From 9 young healthy volunteers (4 women and 5 men) saliva samples were collected either unstimulated by passive drooling or stimulated using a paraffin gum or Salivette® (cotton swab). Saliva volume, protein concentration and salivary protein patterns were analyzed comparatively. RESULTS Samples collected using paraffin gum showed the highest saliva volume (4.1±1.5 ml) followed by Salivette® collection (1.8±0.4 ml) and drooling (1.0±0.4 ml). Saliva protein concentrations (average 1145 μg/ml) showed no significant differences between the three sampling schemes. Each collection approach facilitated the identification of about 160 proteins (≥2 distinct peptides) per subject, but collection-method dependent variations in protein composition were observed. CONCLUSION Passive drooling, paraffin gum and Salivette® each allows similar coverage of the whole saliva proteome, but the specific proteins observed depended on the collection approach. Thus, only one type of collection device should be used for quantitative proteome analysis in one experiment, especially when performing large-scale cross-sectional or multi-centric studies.

δ-Toxin, unlike melittin, has only hemolytic activity and no antimicrobial activity: Rationalization of this specific biological activity

The antimicrobial activity of a synthetic peptide corresponding to δ-hemolysin had been examined. The peptide didnot exhibit antimicrobial activity against gram negative and gram positive micro-organisms unlike other hemolytic peptides like melittin. This lack of antibacterial activity arises due to the inability of δ-hemolysin to perturb the negatively charged bacterial cell surface and permeabilize the bacterial plasma membrane. However, the red blood cell surface has a structure considerably different from bacteria, and does not act as a barrier to molecules reaching the lipid membrane. Hence δ-toxin can lyse erythrocytes. Thus, the specificity in biological activity has been rationalized in terms of differences, in the interaction of the toxin with the bacterial and red blood cell surfaces.

Conformation and activity of δ-lysin and its analogs

Abstract δ-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus . Unlike the bee venom peptide melittin, δ-lysin does not exhibit antibacterial activity. We have synthesized δ-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7–26 and 5–26 of δ-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike δ-lysin, did not self-associate to adopt α-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on δ-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.

A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria.

The development of a mass spectrometric workflow for the sensitive identification and quantitation of the kinetics of changes in metaproteomes, or in particular bacterial pathogens after internalization by host cells, is described. This procedure employs three essential stages: (i) SILAC pulse-chase labeling and infection assay; (ii) isolation of bacteria by GFP-assisted cell sorting; (iii) mass spectrometry-based proteome analysis. This approach displays greater sensitivity than techniques relying on conventional cell sorting and protein separation, due to an efficient combination of a filtration-based purification and an on-membrane digestion. We exemplary describe the use of the workflow for the identification and quantitation of the proteome of 10⁶ cells of Staphylococcus aureus after internalization by S9 human bronchial epithelial cells. With minor modifications, the workflow described can be applied for the characterization of other host-pathogen pairs, permitting identification and quantitation of hundreds of bacterial proteins over a time range of several hours post infection.

Characterization of the EGFR interactome reveals associated protein complex networks and intracellular receptor dynamics.

Growth factor receptor mediated signaling is meanwhile recognized as a complex signaling network, which is initiated by recruiting specific patterns of adaptor proteins to the intracellular domain of epidermal growth factor receptor (EGFR). Approaches to globally identify EGFR‐binding proteins are required to elucidate this network. We affinity‐purified EGFR with its interacting proteins by coprecipitation from lysates of A431 cells. A total of 183 proteins were repeatedly detected in high‐resolution MS measurements. For 15 of these, direct interactions with EGFR were listed in the iRefIndex interaction database, including Grb2, shc‐1, SOS1 and 2, STAT 1 and 3, AP2, UBS3B, and ERRFI. The newly developed Cytoscape plugin ModuleGraph allowed retrieving and visualizing 93 well‐described protein complexes that contained at least one of the proteins found to interact with EGFR in our experiments. Abundances of 14 proteins were modulated more than twofold upon EGFR activation whereof clathrin‐associated adaptor complex AP‐2 showed 4.6‐fold enrichment. These proteins were further annotated with different cellular compartments. Finally, interactions of AP‐2 proteins and the newly discovered interaction of CIP2A could be verified. In conclusion, a powerful technique is presented that allowed identification and quantitative assessment of the EGFR interactome to provide further insight into EGFR signaling.

Activation of the alternative sigma factor SigB of Staphylococcus aureus following internalization by epithelial cells - an in vivo proteomics perspective.

Staphylococcus aureus is a versatile pathogen that can be a commensal but also cause a wide range of different infections. This broad disease spectrum is a reflection of the complex regulation of a large collection of virulence factors that together with metabolic fitness allow adaptation to different niches. The alternative sigma factor SigB is one of the global regulators mediating this adaptation. However, even if SigB contributes to expression of many virulence factors its importance for successful infection greatly varies with the strain and the infection setting analyzed. We have recently established a proteomics workflow that combines high efficiency cell sorting with sensitive mass spectrometry and allows monitoring of global proteome adaptations with roughly one million bacterial cells. Thus, we can now approach the adaptation of pathogens to the intracellular milieu. In the current study this proteomics workflow was used in conjunction with qRT-PCR and confocal fluorescence microscopy to comparatively analyze the adaptation of the S. aureus wild type strain HG001 and its isogenic sigB mutant to the intracellular milieu of human S9 bronchial epithelial cells. The study revealed fast and transient activation of SigB following internalization by human host cells and the requirement of SigB for intracellular growth. Loss of SigB triggered proteome changes reflecting the different residual growth rates of wild type and sigB mutant, respectively, the resistance to methicillin, adaptation to oxidative stress and protein quality control mechanisms.

A proteomic perspective of the interplay of Staphylococcus aureus and human alveolar epithelial cells during infection.

Infectious diseases caused by pathogens such as Staphylococcus aureus are still a major threat for human health. Proteome analyses allow detailed monitoring of the molecular interplay between pathogen and host upon internalization. However, the investigation of the responses of both partners is complicated by the large excess of host cell proteins compared to bacterial proteins as well as by the fact that only a fraction of host cells are infected. In the present study we infected human alveolar epithelial A549 cells with S. aureus HG001 pMV158GFP and separated intact bacteria from host cell debris or infected from non-infected A549 cells by cell sorting to enable detailed proteome analysis. During the first 6.5h in the intracellular milieu S. aureus displayed reduced growth rate, induction of the stringent response, adaptation to microaerobic conditions as well as cell wall stress. Interestingly, both truly infected host cells and those not infected but exposed to secreted S. aureus proteins and host cell factors showed differences in the proteome pattern compared to A549 cells which had never been in contact with S. aureus. However, adaptation reactions were more pronounced in infected compared to non-infected A549 bystander cells.

Bone marrow-derived macrophages from BALB/c and C57BL/6 mice fundamentally differ in their respiratory chain complex proteins, lysosomal enzymes and components of antioxidant stress systems.

UNLABELLED Macrophages are essential components of the innate immune system and crucial for pathogen elimination in early stages of infection. We previously observed that bone marrow-derived macrophages (BMMs) from C57BL/6 mice exhibited increased killing activity against Burkholderia pseudomallei compared to BMMs from BALB/c mice. This effect was particularly pronounced when cells were treated with IFN-γ. To unravel mechanisms that could explain these distinct bactericidal effects, a comparative combined proteome and transcriptome analysis of untreated and IFN-γ treated BALB/c and C57BL/6 BMMs under standardized serum-free conditions was carried out. We found differences in gene expression/protein abundance belonging to cellular oxidative and antioxidative stress systems. Genes/proteins involved in the generation of oxidant molecules and the function of phagosomes (respiratory chain ATPase, lysosomal enzymes, cathepsins) were predominantly higher expressed/more abundant in C57BL/6 BMMs. Components involved in alleviation of oxidative stress (peroxiredoxin, mitochondrial superoxide dismutase) were more abundant in C57BL/6 BMMs as well. Thus, C57BL/6 BMMs seemed to be better equipped with cellular systems that may be advantageous in combating engulfed pathogens. Simultaneously, C57BL/6 BMMs were well protected from oxidative burst. We assume that these variations co-determine differences in resistance between BALB/c and C57BL/6 mice observed in many infection models. BIOLOGICAL SIGNIFICANCE In this study we performed combined transcriptome and proteome analyses on BMMs derived from two inbred mouse strains that are frequently used for studies in the field of host-pathogen interaction research. Strain differences between BALB/c and C57BL/6 BMMs were found to originate mainly from different protein abundance levels rather than from different gene expression. Differences in abundance of respiratory chain complexes and lysosomal proteins as well as differential regulation of components belonging to various antioxidant stress systems help to explain long-known differences between the mouse strains concerning their different susceptibility in several infection models.