Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique

<p>Abstract</p> <p>Background</p> <p><it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by <it>S</it>. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of <it>S</it>. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI™ FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified from mass spectral database searches.</p> <p>Results</p> <p>In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B₁₂, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX.</p> <p>Conclusion</p> <p>Using a multi-step digest approach the LPI™ technique enables the incorporation of a multi-step protease work flow ensuring enough sequence coverage of membrane proteins subsequently leading to the identification of more membrane proteins with higher confidence. Compared to current sub-cellular fractionation procedures and previous published work, the LPI™ technique currently provides the widest coverage of outer membrane proteins identified as demonstrated here for <it>Salmonella </it>Typhimurium.</p

Proteome analysis of serovars Typhimurium and Pullorum of Salmonella enterica subspecies I

BACKGROUND: Salmonella enterica subspecies I includes several closely related serovars which differ in host ranges and ability to cause disease. The basis for the diversity in host range and pathogenic potential of the serovars is not well understood, and it is not known how host-restricted variants appeared and what factors were lost or acquired during adaptations to a specific environment. Differences apparent from the genomic data do not necessarily correspond to functional proteins and more importantly differential regulation of otherwise identical gene content may play a role in the diverse phenotypes of the serovars of Salmonella. RESULTS: In this study a comparative analysis of the cytosolic proteins of serovars Typhimurium and Pullorum was performed using two-dimensional gel electrophoresis and the proteins of interest were identified using mass spectrometry. An annotated reference map was created for serovar Typhimurium containing 233 entries, which included many metabolic enzymes, ribosomal proteins, chaperones and many other proteins characteristic for the growing cell. The comparative analysis of the two serovars revealed a high degree of variation amongst isolates obtained from different sources and, in some cases, the variation was greater between isolates of the same serovar than between isolates with different sero-specificity. However, several serovar-specific proteins, including intermediates in sulphate utilisation and cysteine synthesis, were also found despite the fact that the genes encoding those proteins are present in the genomes of both serovars. CONCLUSION: Current microbial proteomics are generally based on the use of a single reference or type strain of a species. This study has shown the importance of incorporating a large number of strains of a species, as the diversity of the proteome in the microbial population appears to be significantly greater than expected. The characterisation of a diverse selection of strains revealed parts of the proteome of S. enterica that alter their expression while others remain stable and allowed for the identification of serovar-specific factors that have so far remained undetected by other methods

Differential spatiotemporal targeting of Toxoplasma and Salmonella by GBP1 assembles caspase signalling platforms

Human guanylate binding proteins (GBPs), a family of IFNγ-inducible GTPases, promote cell-intrinsic defence against pathogens and host cell death. We previously identified GBP1 as a mediator of cell death of human macrophages infected with Toxoplasma gondii (Tg) or Salmonella Typhimurium (STm). How GBP1 targets microbes for AIM2 activation during Tg infection and caspase-4 during STm infection remains unclear. Here, using correlative light and electron microscopy and EdU labelling of Tg-DNA, we reveal that GBP1-decorated parasitophorous vacuoles (PVs) lose membrane integrity and release Tg-DNA for detection by AIM2-ASC-CASP8. In contrast, differential staining of cytosolic and vacuolar STm revealed that GBP1 does not contribute to STm escape into the cytosol but decorates almost all cytosolic STm leading to the recruitment of caspase-4. Caspase-5, which can bind LPS and whose expression is upregulated by IFNγ, does not target STm pointing to a key role for caspase-4 in pyroptosis. We also uncover a regulatory mechanism involving the inactivation of GBP1 by its cleavage at Asp192 by caspase-1. Cells expressing non-cleavable GBP1D192E therefore undergo higher caspase-4-driven pyroptosis during STm infection. Taken together, our comparative studies elucidate microbe-specific spatiotemporal roles of GBP1 in inducing cell death by leading to assembly and regulation of divergent caspase signalling platforms

Identification and biochemical analysis of a novel APOB mutation that causes autosomal dominant hypercholesterolemia

Patients with autosomal dominant hypercholesterolemia (ADH) have a high risk of developing cardiovascular disease that can be effectively treated using statin drugs. Molecular diagnosis and family cascade screening is recommended for early identification of individuals at risk, but up to 40% of families have no mutation detected in known genes. This study combined linkage analysis and exome sequencing to identify a novel variant in exon 3 of APOB (Arg50Trp). Mass spectrometry established that low-density lipoprotein (LDL) containing Arg50Trp APOB accumulates in the circulation of affected individuals, suggesting defective hepatic uptake. Previously reported mutations in APOB causing ADH have been located in exon 26. This is the first report of a mutation outside this region causing this phenotype, therefore, more extensive screening of this large and highly polymorphic gene may be necessary in ADH families. This is now feasible due to the high capacity of recently available sequencing platforms

Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites

Proteome analysis of bacterial pathogens

The explosive progress over the past decade in the fields of genomics, bioinformatics and mass spectrometry has resulted in an increased capability to investigate and compare the global protein expression of cells, tissues and organisms. The main focus of this study was on characterising the protein expression profiles of different serovars of Salmonella enterica and different serotypeso f Streptococcusp neumoniae in an attempt to identify protein factors associatedw ith host specificity and virulence. A novel approach for typing of bacterial isolates using SELDI TOF MS was developed. A thorough investigation on the effect of different factors on the quality of the SELDI profile was carried out, and the potential of several software programmes to perform cluster analysis of the SELDI data was assessedB. oth cytosolic and membrane-associatepdr oteins were separatedb y 2D GE, and detailed referencem aps of the proteins expressedu nder standardisedc. onditions were created.I n the caseo f Salmonella, it containedm ore than 300 proteins. The comparativea nalysisa t the subspeciesle vel revealedt hat, in many cases,t he variation in the expression patterns was greater between strains with the same serospecificity than betweens erotypes/serovarss, uggestingt hat the serological properties of bacteria do not correlate with differential protein synthesis. However, in the case of Salmonella, where the serovars have different host specificity, the high resolution 2D gel maps revealed several serovar-specific proteins, including enzymes involved in the catabolismo f various substrates,o r in the processo f cell detoxification. Changesi n the expression patterns of the serovars in different growth conditions, such as pH or oxygen availability, were mostly universal amongst the serovars, although a few serovar-specific proteins were also present. The findings revealed parts of the proteome that alter their expression when the microorganism are subjected to unfavourable conditions such as while colonising the host, amongst other parts that remain stable. Overall, the results demonstrated the importance of analysing many different isolates when performing protein expression studies in highly variable microbial populations

p97/VCP targets <i>Toxoplasma gondii</i> vacuoles for parasite restriction in interferon-stimulated human cells

Infection with the parasite Toxoplasma gondii leads to production of interferon gamma (IFNγ) that stimulates cells to upregulate defense proteins targeting the parasite for cell intrinsic elimination or growth restriction. Various host defense mechanisms operate at the parasitophorous vacuole (PV) in different human cell types leading to PV disruption, acidification, or membrane envelopment. Ubiquitin and p62 are players in all human host control mechanisms of Toxoplasma, but other unifying proteins have not been identified. Here, we show that p97/valosin-containing protein (VCP), as well as its associated proteins ANKRD13A and UBXD1 control Toxoplasma infection while recruited to the PV in IFNγ-stimulated endothelial cells. Convergent deposition of ANKRD13A, p97/VCP, and UBXD1 onto the same vacuole is dependent on vacuolar ubiquitination and observed within 2 h post-infection. ANKRD13A, p97/VCP, and UBXD1 all drive the acidification mechanism of the vacuole, which is the IFNγ-dependent control pathway of Toxoplasma in endothelial cells. We assessed p97/VCP in Toxoplasma control in various human cells and demonstrate that p97/VCP is a universal IFNγ-dependent host restriction factor targeting the Toxoplasma PV in epithelial (HeLa) and endothelial cells (human umbilical vein endothelial cells), fibroblasts (human foreskin fibroblast), and macrophages (THP1).</p