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

The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration

Background The most commonly used diagnostic tool for soil-transmitted helminths (STH) is the Kato-Katz (KK) thick smear technique. However, numerous studies have suggested that the sensitivity of KK can be problematic, especially in low prevalence and low intensity settings. An emerging alternative is quantitative polymerase chain reaction (qPCR). Methods In this study, both KK and qPCR were conducted on stool samples from 648 participants in an STH epidemiology study conducted in the delta region of Myanmar in June 2016. Results Prevalence of any STH was 20.68% by KK and 45.06% by qPCR. Prevalence of each individual STH was also higher by qPCR than KK, the biggest difference was for hookworm with an approximately 4-fold increase between the two diagnostic techniques. Prevalence of Ancylostoma ceylanicum, a parasite predominately found in dogs, was 4.63%, indicating that there is the possibility of zoonotic transmission in the study setting. In individuals with moderate to high intensity infections there is evidence for a linear relationship between eggs per gram (EPG) of faeces, derived from KK, and DNA copy number, derived from qPCR which is particularly strong for Ascaris lumbricoides. Conclusions The use of qPCR in low prevalence settings is important to accurately assess the epidemiological situation and plan control strategies for the ‘end game’. However, more work is required to accurately assess STH intensity from qPCR results and to reduce the cost of qPCR so that is widely accessible in STH endemic countries.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data

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 ‘pooling approach’ can yield a low frequency of ‘missed’ 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 ‘pools-of-five’. 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.Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

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

Ethnicity and COVID-19 cardiovascular complications: a multi-center UK cohort

BACKGROUND: Recent reports suggest an association between ethnicity and COVID-19 mortality. In the present multi-center study, we aimed to assess the differences underlying this association, and ascertain whether ethnicity also mediates other aspects of COVID-19 like cardiovascular complications. METHODS: Data were collected from a mixed-ethnicity UK cohort of 613 patients admitted and diagnosed COVID-19 positive, across six hospitals in London during the second half of March 2020: 292 were White Caucasian ethnicity, 203 were Asian and 118 were of Afro-Caribbean ethnicity. RESULTS: Caucasian patients were older (P<0.001) and less likely to have hypertension (P=0.038), while Afro-Caribbean patients had higher prevalence of diabetes mellitus (P<0.001). Asian patients were more likely to present with venous thromboembolic disease (adj.OR=4.10, 95% CI 1.49-11.27, P=0.006). On the other hand, Afro-Caribbean had more heart failure (adj.OR=3.64, 95% CI 1.50-8.84, P=0.004) and myocardial injury (adj.OR=2.64, 95% CI 1.10-6.35, P=0.030). Importantly, our adjusted multi-variate Cox regression analysis revealed significantly higher all-cause mortality both for Asian (adj.HR=1.89, 95% CI 1.23-2.91, P=0.004) and Afro-Caribbean ethnicity (adj.HR=2.09, 95% CI 1.30-3.37, P=0.002). CONCLUSIONS: Our data show that COVID-19 may have different presentations and follow different clinical trajectories depending on the ethnicity of the affected subject. Awareness of complications more likely to arise in specific ethnicities will allow a more timely diagnosis and preventive measures for patients at risk. Due to increased mortality, individuals of Afro-Caribbean and Asian ethnicity should be considered as high-risk groups. This may have an impact on health-resource allocation and planning, definition of vulnerable groups, disease management, and the protection of healthcare workers at the frontline

High sensitivity troponin and COVID-19 outcomes

Background: Recent reports have demonstrated high troponin levels in patients affected with COVID-19. In the present study, we aimed to determine the association between admission and peak troponin levels and COVID-19 outcomes. / Methods: This was an observational multi-ethnic multi-centre study in a UK cohort of 434 patients admitted and diagnosed COVID-19 positive, across six hospitals in London, UK during the second half of March 2020. / Results: Myocardial injury, defined as positive troponin during admission was observed in 288 (66.4%) patients. Age (OR: 1.68 [1.49–1.88], p <.001), hypertension (OR: 1.81 [1.10–2.99], p =.020) and moderate chronic kidney disease (OR: 9.12 [95% CI: 4.24–19.64], p <.001) independently predicted myocardial injury. After adjustment, patients with positive peak troponin were more likely to need non-invasive and mechanical ventilation (OR: 2.40 [95% CI: 1.27–4.56], p =.007, and OR: 6.81 [95% CI: 3.40–13.62], p <.001, respectively) and urgent renal replacement therapy (OR: 4.14 [95% CI: 1.34–12.78], p =.013). With regards to events, and after adjustment, positive peak troponin levels were independently associated with acute kidney injury (OR: 6.76 [95% CI: 3.40–13.47], p <.001), venous thromboembolism (OR: 11.99 [95% CI: 3.20–44.88], p <.001), development of atrial fibrillation (OR: 10.66 [95% CI: 1.33–85.32], p =.026) and death during admission (OR: 2.40 [95% CI: 1.34–4.29], p =.003). Similar associations were observed for admission troponin. In addition, median length of stay in days was shorter for patients with negative troponin levels: 8 (5–13) negative, 14 (7–23) low-positive levels and 16 (10–23) high-positive (p <.001). / Conclusions: Admission and peak troponin appear to be predictors for cardiovascular and non-cardiovascular events and outcomes in COVID-19 patients, and their utilisation may have an impact on patient management

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

Identification of protective antigens for vaccination against systemic salmonellosis

There is an urgent medical need for improved vaccines with broad serovar coverage and high efficacy against systemic salmonellosis. Subunit vaccines offer excellent safety profiles but require identification of protective antigens, which remains a challenging task. Here, I review crucial properties of Salmonella antigens that might help to narrow down the number of potential candidates from more than 4000 proteins encoded in Salmonella genomes, to a more manageable number of 50-200 most promising antigens. I also discuss complementary approaches for antigen identification and potential limitations of current pre-clinical vaccine testing