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

Pooling as a Strategy for the Timely Diagnosis of Soil-Transmitted Helminths in Stool: Value and Reproducibility

Background: The strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low- and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy. Results: The sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a \u27pooling approach\u27 can yield a low frequency of \u27missed\u27 infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools. Conclusions: Our results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in \u27pools-of-five\u27. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method

Characterisation of the outer membrane proteome of Salmonella Spp.

Despite successful sequencing and subsequent comparison of Salmonella Typhimurium (S. Typhimurium) and Salmonella Typhi (S. Typhi) genomes, mechanisms driving S. Typhi host specificity and pathogenicity remain unclear. The characterisation of the surface proteomes of the two serotypes may reveal proteins that play a role in host pathogen interaction, thereby permitting S. Typhi to establish persistent infections. Surface proteins represent a small fraction of the total cell and are difficult to extract preferentially. This study evaluated a range of methods for the extraction of outer membrane proteins (OMPs) in S. Typhimurium, including previously published protocols based on differential solubilisation and a novel approach based on the LPI™ FlowCell. Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified these proteins, and their association with the outer membrane confirmed against a database containing in silica predictions of the sub-cellular location of all ORFs in both genomes. This comprehensive approach identified 73 OMPs, representing 63% of all predicted S. Typhimurium aMPs. When applied to S. Typhi, several OMPs undetected in S. Typhimurium extracts were identified including proteins involved in capsule synthesis. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) detected OMPs potentially upregulated in S. Typhi, resulting in the identification of 20 upregulated spots containing 15 OMPs, five previously undetected. OMPs with various functions were identified, including porins, receptor proteins, transporters and enzymes. Several OMPs with potential roles in virulence, such as the two regulators SlyB and YfgL modulati ng the expression of pathogenicity islands-encoded proteins, were detected. Additionally, the TolC protein previously implicated in antibiotic resistance and invasion as part of the AcrAB-ToIC efflux system was present in both serotypes. Other examples include the FepA and IroN, involved in iron uptake, potentially upregulated in S. Typhi. The expression of many hypothetical aMPs with poorly defined functions was also confirmed, providing useful targets for future studies.EThOS - Electronic Theses Online ServiceGBUnited Kingdo