Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium.

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.

Growth-restricting effects of siRNA transfections: a largely deterministic combination of off-target binding and hybridization-independent competition.

Perturbation of gene expression by means of synthetic small interfering RNAs (siRNAs) is a powerful way to uncover gene function. However, siRNA technology suffers from sequence-specific off-target effects and from limitations in knock-down efficiency. In this study, we assess a further problem: unintended effects of siRNA transfections on cellular fitness/proliferation. We show that the nucleotide compositions of siRNAs at specific positions have reproducible growth-restricting effects on mammalian cells in culture. This is likely distinct from hybridization-dependent off-target effects, since each nucleotide residue is seen to be acting independently and additively. The effect is robust and reproducible across different siRNA libraries and also across various cell lines, including human and mouse cells. Analyzing the growth inhibition patterns in correlation to the nucleotide sequence of the siRNAs allowed us to build a predictor that can estimate growth-restricting effects for any arbitrary siRNA sequence. Competition experiments with co-transfected siRNAs further suggest that the growth-restricting effects might be linked to an oversaturation of the cellular miRNA machinery, thus disrupting endogenous miRNA functions at large. We caution that competition between siRNA molecules could complicate the interpretation of double-knockdown or epistasis experiments, and potential interactions with endogenous miRNAs can be a factor when assaying cell growth or viability phenotypes.

Factors associated with relevant knowledge of intestinal schistosomiasis and intention to participate in treatment campaigns: a cross sectional survey among school children at Ijinga Island on Lake Victoria, North-Western Tanzania.

BACKGROUND: Annual Mass Drug Administration (MDA) using praziquantel targeting primary school children is the main control strategy against schistosomiasis in Tanzania. However, there are concerns about decreasing participation in mass drug administration among primary school children for unknown reasons. Therefore, the aim of this study was to identify factors related to relevant knowledge about schistosomiasis and the intention to participate in mass drug administration among primary school children in order to give recommendations for future projects. METHODS: A cross sectional, extended knowledge, attitudes and practices (KAP) survey was conducted among 356 primary school children aged 5-17 years in February-March 2016 using a pre-tested questionnaire. This survey was part of a baseline assessment for an integrated proof of concept study aiming towards schistosomiasis elimination on Ijinga Island. Outcomes of interest in logistic regression analysis were relevant knowledge and high intention to participate in treatment campaigns. Explanatory variables were sociodemographic information sources and elements aligned to Protection Motivation Theory (PMT). RESULTS: Only 17% of the children had relevant intestinal schistosomiasis related knowledge and very few of them knew any of the S. mansoni manifestations and complications. Factors associated with relevant schistosomiasis knowledge were previous diagnosis of schistosomiasis (aOR = 2.43, 95%CI: 1.1-5.6), having heard about schistosomiasis at school (aOR = 9.94, 95%CI: 5.0-19.7) and being enrolled in 6th or 7th grade (aOR = 3.94, 95%CI: 1.3-11.8). Only 40% of the children demonstrated high intention to participate in treatment campaigns. Factors associated with high intention to participate in MDA were previous diagnosis (aOR = 2.23, 95%CI: 1.1-4.7), perceived general risk of disease transmission by lake water (aOR = 1.79, 95%CI: 1.0-3.1), perceived own vulnerability of getting infected (aOR = 5.10, 95%CI: 2.1-12.6), perceived danger of the disease (aOR = 2.47, 95%CI: 1.3-4.8) and the perceived effectiveness of medicaments to cure the disease (aOR = 2.86, 95%CI: 1.4-5.7). CONCLUSIONS: The minority of the school children had high level of theoretical knowledge about schistosomiasis and a small proportion of the children demonstrated high intention to participate in mass drug administration. In general, practical knowledge on preventive measures such as taking anti-schistosomiasis drug during MDA need to be impacted in school children to increase their participation in the control program.

Virulence function of the Ustilago maydis sterol carrier protein 2.

The peroxisomal sterol carrier protein 2 (Scp2) of the biotrophic maize pathogen Ustilago maydis was detected in apoplastic fluid, suggesting that it might function as a secreted effector protein. Here we analyze the role of the scp2 gene during plant colonization. We used reverse genetics approaches to delete the scp2 gene, determined stress sensitivity and fatty acid utilization of mutants, demonstrated secretion of Scp2, used quantitative reverse transcription polymerase chain reaction for expression analysis and expressed GFP-Scp2 fusion proteins for protein localization. scp2 mutants were strongly attenuated in virulence and this defect manifested itself during penetration. Scp2 localized to peroxisomes and peroxisomal targeting was necessary for its virulence function. Deletion of scp2 in U. maydis interfered neither with growth nor with peroxisomal ß-oxidation. Conventionally secreted Scp2 protein could not rescue the virulence defect. scp2 mutants displayed an altered localization of peroxisomes. Our results show a virulence function for Scp2 during penetration that is probably carried out by Scp2 in peroxisomes. We speculate that Scp2 affects the lipid composition of membranes and in this way ensures the even cellular distribution of peroxisomes.

Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens.

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended "on-target" mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by "off-target" effects resulting from partial complementarity of siRNAs to multiple mRNAs via the "seed" region (i.e., nucleotides 2-8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying "seed reagents" for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens.

Simultaneous analysis of large-scale RNAi screens for pathogen entry.

BACKGROUND: Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries. RESULTS: We show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied. CONCLUSIONS: Our results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field.

Near surface swimming of Salmonella Typhimurium explains target-site selection and cooperative invasion.

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5-3 s in "near surface swimming". This increased the local pathogen density and facilitated "scanning" of the host surface topology. We observed transient TTSS-1 and fim-independent "stopping" and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria.

Cultural adaption and validation of the Explanatory Model Interview Catalogue-Community Stigma Scale in the assessment of public stigma related to schistosomiasis in lakeshore areas of Mwanza region, Tanzania.

BACKGROUND: Previous qualitative studies on attitudes towards schistosomiasis demonstrated inconclusive results on the extent of stigma towards schistosomiasis in endemic communities around the world. The Explanatory Model Interview Catalogue-Community Stigma Scale (EMIC-CSS) has been used and validated for the assessment of public stigma across numerous countries in various health conditions. This study tested the performance of the scale in the context of stigma related to schistosomiasis in twelve communities in the three districts of Magu, Nyamagana and Ilemela in Mwanza region, Tanzania. METHODOLOGY/PRINCIPAL FINDINGS: The 15-item-version of the EMC-CSS was first translated to Kiswahili language. The translation was discussed within the research team to retain the meaning of the items and implement cultural adaptations. Validation of the adapted EMIC-CSS scale was conducted following the framework of Herdman and Fox- Rushby. A pilot study with 41 participants from two communities provided the basis for testing the performance of each item and assessing the semantic and operational equivalence of the scales. In addition, eight qualitative focus group discussions (FGDs) were conducted to evaluate the conceptional equivalence of the EMIC-CSS. Finally, the performance of the adjusted scale was tested on 200 participants with a 50:50 male-female ratio from ten communities. The mean score of the EMIC-CSS M = 8.35 (SD = 6.63) shows clear indications for public stigma towards schistosomiasis. The EMIC-CSS demonstrated a good internal consistency with Cronbach's alpha α = .857 and no floor and ceiling effects. CONCLUSION/SIGNIFICANCE: The results demonstrate that the EMIC-CSS is a useful instrument in assessing public stigma towards schistosomiasis and allow a clear recommendation of the EMIC-CSS for schistosomiasis in the Tanzanian culture. However, future studies are additionally recommended to address specific aspects and forms of the disease and how they contribute to the development of stigma towards schistosomiasis.

A cluster randomized trial for improving mental health and well-being of persons affected by leprosy or buruli ulcer in Nigeria: A study protocol.

This protocol describes a study in which we would assess the effect of using community lay counselors, self-help groups (SHGs), and trained frontline health workers to reduce mental disorders and improve quality of life (QOL) of persons affected by leprosy or Buruli ulcer (BU). A cluster randomized controlled study design will be employed. The study will involve persons affected by leprosy or BU. Ten local government areas (clusters) with the highest number of notified leprosy or BU cases between 2014 and 2018 in Southern Nigeria will be purposively selected. The clusters will be randomized into intervention and control groups using a computer-generated list of random numbers. At baseline, data were collected using the following validated questionnaires, Patient Health Questionnaire, Generalized Anxiety Disorder questionnaire, Stigma Assessment and Reduction of Impact Scale, World Health Organization QOL BREF and Warwick-Edinburgh Mental Well-being scale among persons affected by leprosy or BU. The intervention will last for 2 years and will involve use of community lay counselors, SHGs, and appropriately trained frontline health workers in reducing mental disorders and improving QOL of persons affected by leprosy or BU. This project postulates that the reduction of burden of mental health problems and improved QOL among persons affected by leprosy or BU could be achieved through a holistic approach involving SHGs, appropriately trained community opinion leaders, and general health-care workers as well as a functional referral system. If successful, the model will be integrated into the activities of the National Tuberculosis and Leprosy Control Programme and scaled up nationwide. Trial registration: ISRCTN Registry: ISRCTN 83649248. https://trialsearch. who.int/Trial2.aspx? TrialID % ISRCTN83649248 Prospectively registered.

Salmonella enterica serovar Typhimurium binds to HeLa cells via Fim-mediated reversible adhesion and irreversible type three secretion system 1-mediated docking.

The food-borne pathogen Salmonella enterica serovar Typhimurium invades mammalian epithelial cells. This multistep process comprises bacterial binding to the host cell, activation of the Salmonella type three secretion system 1 (T1), injection of effector proteins, triggering of host cell actin rearrangements, and S. Typhimurium entry. While the latter steps are well understood, much less is known about the initial binding step. Earlier work had implicated adhesins (but not T1) or T1 (but not other adhesins). We have studied here the Salmonella virulence factors mediating S. Typhimurium binding to HeLa cells. Using an automated microscopy assay and isogenic S. Typhimurium mutants, we analyzed the role of T1 and of several known adhesins (Fim, Pef, Lpf, Agf, and Shd) in host cell binding. In wild-type S. Typhimurium, host cell binding was mostly attributable to T1. However, in the absence of T1, Fim (but not Pef, Lpf, Agf, and Shd) also mediated HeLa cell binding. Furthermore, in the absence of T1 and type I fimbriae (Fim), we still observed residual binding, pointing toward at least one additional, unidentified binding mechanism. Dissociation experiments established that T1-mediated binding was irreversible ("docking"), while Fim-mediated binding was reversible ("reversible adhesion"). Finally, we show that noninvasive bacteria docking via T1 or adhering via Fim can efficiently invade HeLa cells, if actin rearrangements are triggered in trans by a wild-type S. Typhimurium helper strain. Our data show that binding to HeLa cells is mediated by at least two different mechanisms and that both can lead to invasion if actin rearrangements are triggered.

Measuring endemicity and burden of leprosy across countries and regions: A systematic review and Delphi survey.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, the annual new case detection in 2019 was 202,189 globally. Measuring endemicity levels and burden in leprosy lacks a uniform approach. As a result, the assessment of leprosy endemicity or burden are not comparable over time and across countries and regions. This can make program planning and evaluation difficult. This study aims to identify relevant metrics and methods for measuring and classifying leprosy endemicity and burden at (sub)national level. METHODS: We used a mixed-method approach combining findings from a systematic literature review and a Delphi survey. The literature search was conducted in seven databases, searching for endemicity, burden and leprosy. We reviewed the available evidence on the usage of indicators, classification levels, and scoring methods to measure and classify endemicity and burden. A two round Delphi survey was conducted to ask experts to rank and weigh indicators, classification levels, and scoring methods. RESULTS: The literature review showed variation of indicators, levels, and cut-off values to measure leprosy endemicity and/or burden. The most used indicators for endemicity include new case detection rate (NCDR), new cases among children and new cases with grade 2 disability. For burden these include NCDR, MB cases, and prevalence. The classification levels 'high' and 'low' were most important. It was considered most relevant to use separate scoring methods for endemicity and burden. The scores would be derived by use of multiple indicators. CONCLUSION: There is great variation in the existing method for measuring endemicity and burden across countries and regions. Our findings contribute to establishing a standardized uniform approach to measure and classify leprosy endemicity and burden at (sub)national level, which would allow effective communication and planning of intervention strategies.

The yield of Auramine O staining using led microscopy with bleach treated sputum samples for detection of pulmonary tuberculosis at St. Peter tuberculosis specialized hospital, Addis Ababa, Ethiopia.

BACKGROUND: Smear microscopy is the mainstay for diagnosis of Tuberculosis (TB) in Ethiopia. This technique; however, is insensitive to detect Mycobacteria from most clinical specimens. Currently, light emitting diode (LED) fluorescence microscope is advocated to be used in high Tuberculosis (TB) burden settings by World Health Organization (WHO). However, the utility of this method is not evaluated for bleach treated sputum samples in Ethiopia. OBJECTIVE: The objective of the study is to evaluate the diagnostic importance of Auramine O (AO) staining in direct and concentrated sputum against conventional Zehil-Neelsen (ZN) and culture from the sputum samples of suspected pulmonary tuberculosis patients. METHODS: A cross-sectional study was conducted on 346 adult new pulmonary TB suspected patients at St. Peter's Specialized Hospital, Addis Ababa, Ethiopia. Three sputum samples (spot-morning-spot) were collected in sterile cups for direct Zehil-Neelsen and AO staining. Morning sputum samples were used for Mycobacterial culture on Mycobacterial Growth Indicator Tube (MGIT) 960. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated against the gold standard culture method. Data were analyzed using STATA version 13.0. All statistical tests were considered as statistically significant if the two sided P-value was < 0.05. RESULTS: Bleach treated sputum samples with AO staining yielded more cases as compared to direct ZN and direct AO by 6.3% and 11.5%, respectively. The sensitivity of concentrated AO and direct AO were remarkably high as compared to conventional ZN (71.8% vs. 44.5% and 62.7% vs. 44.5%). The concentrated sputum with staining of AO had a high rate (18.6%) of detecting scanty graded smears as compared to conventional ZN method. CONCLUSIONS: Our findings indicated that the concentrated sputum with AO staining yielded high rate of sensitivity (71.8%) as compared to the conventional ZN method (44.5%). Moreover, the concentrated sputum with AO staining had superior ability in detecting scanty graded smears compared to the conventional ZN method. Therefore, it is recommended to utilize AO staining with LED microscopy for better diagnosis of Acid Fast Bacilli (AFB) from TB suspected cases and patients with pauci-bacillary TB in Ethiopia.

Multi-omic measurements of heterogeneity in HeLa cells across laboratories.

Reproducibility in research can be compromised by both biological and technical variation, but most of the focus is on removing the latter. Here we investigate the effects of biological variation in HeLa cell lines using a systems-wide approach. We determine the degree of molecular and phenotypic variability across 14 stock HeLa samples from 13 international laboratories. We cultured cells in uniform conditions and profiled genome-wide copy numbers, mRNAs, proteins and protein turnover rates in each cell line. We discovered substantial heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto varieties, and observed progressive divergence within a specific cell line over 50 successive passages. Genomic variability has a complex, nonlinear effect on transcriptome, proteome and protein turnover profiles, and proteotype patterns explain the varying phenotypic response of different cell lines to Salmonella infection. These findings have implications for the interpretation and reproducibility of research results obtained from human cultured cells.

Experimental approaches to phenotypic diversity in infection.

Microbial infections are burdening human health, even after the advent of antibiotics, vaccines and hygiene. Thus, infection biology has aimed at the molecular understanding of the pathogen-host interaction. This has revealed key virulence factors, host cell signaling pathways and immune responses. However, our understanding of the infection process is still incomplete. Recent evidence suggests that phenotypic diversity can have important consequences for the infection process. Diversity arises from the formation of distinct subpopulations of pathogen cells (with distinct virulence factor expression patterns) and host cells (with distinct response capacities). For technical reasons, such phenotypic diversity has often been overlooked. We are highlighting several striking examples and discuss the experimental approaches available for analyzing the different subpopulations. Single cell reporters and approaches from systems biology do hold much promise.

Autophagy Proteins Promote Repair of Endosomal Membranes Damaged by the Salmonella Type Three Secretion System 1.

Salmonella Typhimurium (S.Tm) is an enteropathogen requiring multiple virulence factors, including two type three secretion systems (T1 and T2). T1 triggers epithelium invasion in which the bacteria are taken up into endosomes that mature into Salmonella-containing vacuoles (SCV) and trigger T2 induction upon acidification. Mechanisms controlling endosome membrane integrity or pathogen egress into the cytosol are incompletely understood. We screened for host factors affecting invasion and SCV maturation and identified a role for autophagy in sealing endosomal membranes damaged by T1 during host cell invasion. S.Tm-infected autophagy-deficient (atg5(-/-)) cells exhibit reduced SCV dye retention and lower T2 expression but no effects on steps preceding SCV maturation. However, in the absence of T1, autophagy is dispensable for T2 induction. These findings establish a role of autophagy at early stages of S.Tm infection and suggest that autophagy-mediated membrane repair might be generally important for invasive pathogens and endosomal membrane function.

gespeR: a statistical model for deconvoluting off-target-confounded RNA interference screens.

Small interfering RNAs (siRNAs) exhibit strong off-target effects, which confound the gene-level interpretation of RNA interference screens and thus limit their utility for functional genomics studies. Here, we present gespeR, a statistical model for reconstructing individual, gene-specific phenotypes. Using 115,878 siRNAs, single and pooled, from three companies in three pathogen infection screens, we demonstrate that deconvolution of image-based phenotypes substantially improves the reproducibility between independent siRNA sets targeting the same genes. Genes selected and prioritized by gespeR are validated and shown to constitute biologically relevant components of pathogen entry mechanisms and TGF-ß signaling. gespeR is available as a Bioconductor R-package.

Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis.

The demand on the biotechnological production of proteins for pharmaceutical, medical and industrial applications is steadily growing. For the production of challenging proteins, we aim to establish a novel expression platform in the well characterized eukaryotic microorganism Ustilago maydis. In filaments of this fungus, secretion of the endochitinase Cts1 depends on mRNA transport along microtubules, which is mediated by the key RNA-binding protein Rrm4. Here, we report two important findings: (i) Cts1 secretion occurs via a novel unconventional route and (ii) this secretory mechanism can be exploited for the export of active heterologous proteins. Initially, we used ß-glucuronidase (Gus) as a reporter for unconventional secretion. This bacterial enzyme is inactivated by N-glycosylation during its passage through the conventional eukaryotic secretory pathway. By contrast, in our system Gus was exported in its active form by fusion to Cts1 confirming its secretion by an unconventional route. As a proof-of-principle for economically important biopharmaceuticals we expressed an active single-chain antibody. Importantly, the novel protein export pathway circumvents N-glycosylation which is advantageous in many applications, e.g., to avoid undesired immune reactions in humans. Thus, the unconventional Cts1 secretion machinery has a high potential for the production of biotechnologically relevant proteins.