Selection of Salmonella enterica Serovar Typhi Genes Involved during Interaction with Human Macrophages by Screening of a Transposon Mutant Library

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 105 transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated

Familial aggregation and heritability of Wuchereria bancrofti infection

Background. The familial recurrence risk of lymphatic filariasis (LF) is unknown. This case study aimed to evaluate the familial susceptibility to infection with Wuchereria bancrofti and to microfilaremia in a village of the Republic of Congo. Methods. The heritability and intrafamilial correlation coefficients were assessed for both W bancrofti infection and microfilaremia by controlling for individual risk factors, environmental influence, and household effects. Results. Pedigree charts were constructed for 829 individuals, including 143 individuals with a diagnosis of W bancrofti circulating filarial antigens (CFAs) and 44 who also had microfilariae (MF). There was no intrafamilial correlation regarding CFA levels. However, the presence of MF (rho = 0.45) and microfilarial density (rho = 0.44) were significantly correlated among parent-offspring pairs. Heritability estimates for CFA positivity and intensity were 0.23 and 0.18, respectively. Heritability estimates were high for microfilarial positivity (h(2) = 0.74) and microfilarial density traits (h(2) = 0.81). Conclusions. Our study suggests that the acquisition of LF is mainly driven by environmental factors and habits and that genetic factors are moderately involved in the regulation of infection. By contrast, genetic factors play a major role in both the presence and intensity of microfilaremia

Uptake and intracellular survival of mutants within macrophages during the mixed infection assay.

<p>Competitive index (CI) assays for uptake (0 h) and intracellular survival (24 h post-infection) against the nalidixic acid-resistant wild-type <i>S</i>. Typhi (DEF566) were performed for all 28 isogenic mutants during infection of cultured THP-1 human macrophages. Functional classes are indicated below the gene names. Data presented are the mean ± standard error of the mean of at least three independent experiments performed in duplicate. Asterisks (*) represent CI values for mutants which are significantly different from 1 (<i>P</i><0.05).</p

Functional classification of 130 <i>S</i>. Typhi genes identified following competitive selection in macrophages.

<p>Functional classes are indicated on the left and the number of genes within a class is indicated in bold on the right of each bar. The number of mutants created among each class is shown in parentheses on the left of each class.</p

Effect of plasmid-borne gene complementation on <i>S</i>. Typhi deletion mutants during competitive assays within macrophages.

a<p>CI, competitive index.</p>b<p>The wild-type counterpart is represented by the nalidixic acid-resistant wild-type <i>S</i>. Typhi (DEF566).</p>c<p>Complemented mutants all carry the low-copy-number cloning vector pWSK29 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036643#pone.0036643-Wang1" target="_blank">[36]</a> harbouring the respective deleted gene or gene cluster.</p>d<p>Data presented are the mean ± standard error of the mean of at least three independent experiments performed in duplicate, where the deletion mutants were mixed either with the wild-type strain or the respective complemented mutant during infection of human macrophages. Asterisks (*) represent CI values for mutants which are significantly different from 1 (<i>P</i><0.05).</p

Summary of <i>S</i>. Typhi deletion mutants.

a<p>Loci which have been inactivated in each of the 28 markerless deletion mutants created for this study are listed. Numbers or characters in bold among a gene cluster represent ORF(s) or genes selected following initial screening through macrophages.</p>b<p>Log₂ of output/input values for genes identified following screening of mutant pool through macrophages. For gene clusters, the fold-changes are associated to selected genes (bold) among the cluster.</p>c<p>Phenotypes have been deduced from results of competitive assays in combination with those from individual infections performed in this study using <i>S</i>. Typhi deletion mutants, the wild-type strain, and the nalidixic acid-resistant wild-type strain (DEF566).</p>d<p>Gene or ORF products which are among <i>S</i>. Typhi antigens detected in blood of typhoid fever patients according to previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036643#pone.0036643-Harris1" target="_blank">[56]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036643#pone.0036643-Hu1" target="_blank">[58]</a>.</p>e<p>Percentages represent mutant swimming diameter in mm divided by that of the wild-type, thus indicating the level of motility remaining for the mutants in comparison to the wild-type. Results represent the mean ± standard error of the mean (SEM) of at least three independent experimental replicates and Student's two-tailed <i>t</i>-test was used for statistical analysis.</p>f<p>Values were obtained by subtracting mutant inhibition diameter in mm by that of the wild-type. Results represent the mean ± SEM of at least two independent experimental replicates and Student's two-tailed <i>t</i>-test was used for statistical analysis.</p>g<p>Results for mutant are significantly different from those of the wild-type (<i>P</i><0.05).</p>h<p>ND, no defects during interaction with macrophages are reported in previously published literature on <i>Salmonella</i> serovars other than Typhi.</p>i<p>Pseudogene in <i>S</i>. Typhi strains CT18 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036643#pone.0036643-Parkhill1" target="_blank">[66]</a> and Ty2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036643#pone.0036643-Deng1" target="_blank">[99]</a>.</p>j<p>Unique to <i>S</i>. Typhi compared to <i>S</i>. Typhimurium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036643#pone.0036643-Parkhill1" target="_blank">[66]</a>.</p>k<p>Motility assay was done at 37°C for Δ<i>typA</i>, instead of 30°C used for other mutants, since this mutant exhibited reduced <i>in vitro</i> growth at 30°C (data not shown).</p

Uptake and intracellular survival rates of mutants tested individually in macrophages.

<p>THP-1 human macrophages were infected with <i>S</i>. Typhi wild-type strain ISP1820, all 28 isogenic mutants, and the complemented Δ<i>acrA</i>(pWSK<i>acrA</i>) strain. The number of intracellular bacteria was determined upon uptake (0 h) and during survival (24 h post-infection) within macrophages. Functional classes are indicated below the gene names. The values for percent recovery were normalized to the wild-type control value, defined as 100% at each time point. Data presented are the mean ± standard error of the mean of at least three independent experiments performed in duplicate. Asterisks (*) represent percentages for mutants which are significantly different from the isogenic wild-type (<i>P</i><0.05).</p

Familial aggregation and heritability of Loa loa microfilaremia

Background. For a given prevalence of Loa loa microfilaremia, the proportion of people with high densities varies significantly between communities. We hypothesized that this variation is related to the existence of familial clusters of hypermicrofilaremic individuals that would be the consequence of a genetic predisposition to present high L. loa microfilarial densities. Methods. A familial study was performed in 10 villages in the Okola Health District of Cameroon. Intrafamilial correlation coefficients and heritability estimates were assessed for both the presence of L. loa microfilaremia and individual microfilarial densities by controlling for age, sex, Mansonella perstans coinfection, and household effects. Results. Pedigrees were constructed for 1126 individuals. A significant familial susceptibility to be microfilaremic for L. loa was found for first-degree relatives (rho = 0.08, P<.05; heritability = 0.23). Regarding individual microfilarial densities, a significant familial aggregation was demonstrated (rho = 0.36 for first-degree and 0.27 for second-degree relatives). For first-degree relatives, the highest coefficient was found between mothers and daughters (rho = 0.57). The overall heritability estimate for L. loa microfilarial density was 0.24 (P=.003). Conclusions. A significant genetic component governs L. loa microfilarial density. This supports the hypothesis that a genetic predisposition to be hypermicrofilaremic exists, leading to the presence of familial clusters of individuals at risk for postivermectin severe adverse events. This finding should be taken into account while developing sampling strategies (including a household-level sampling) to identify villages where community-directed treatment with ivermectin cannot be applied

Prognosis of poorly cohesive gastric cancer after complete cytoreductive surgery with or without hyperthermic intraperitoneal chemotherapy (CYTO-CHIP study)

International audienceAbstract Background The incidence of gastric poorly cohesive carcinoma (PCC) is increasing. The prognosis for patients with peritoneal metastases remains poor and the role of cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) is controversial. The aim was to clarify the impact of gastric PCC with peritoneal metastases treated by CRS with or without HIPEC. Methods All patients with peritoneal metastases from gastric cancer treated with CRS with or without HIPEC, in 19 French centres, between 1989 and 2014, were identified from institutional databases. Clinicopathological characteristics and outcomes were compared between PCC and non-PCC subtypes, and the possible benefit of HIPEC was assessed. Results In total, 277 patients were included (188 PCC, 89 non-PCC). HIPEC was performed in 180 of 277 patients (65 per cent), including 124 of 188 with PCC (66 per cent). Median overall survival (OS) was 14.7 (95 per cent c.i. 12.7 to 17.3) months in the PCC group versus 21.2 (14.7 to 36.4) months in the non-PCC group (P &lt; 0.001). In multivariable analyses, PCC (hazard ratio (HR) 1.51, 95 per cent c.i. 1.01 to 2.25; P = 0.044) was associated with poorer OS, as were pN3, Peritoneal Cancer Index (PCI), and resection with a completeness of cytoreduction score of 1, whereas HIPEC was associated with improved OS (HR 0.52; P &lt; 0.001). The benefit of CRS-HIPEC over CRS alone was consistent, irrespective of histology, with a median OS of 16.7 versus 11.3 months (HR 0.60, 0.39 to 0.92; P = 0.018) in the PCC group, and 34.5 versus 14.3 months (HR 0.43, 0.25 to 0.75; P = 0.003) in the non-PCC group. Non-PCC and HIPEC were independently associated with improved recurrence-free survival and fewer peritoneal recurrences. In patients who underwent HIPEC, PCI values of below 7 and less than 13 were predictive of OS in PCC and non-PCC populations respectively. Conclusion In selected patients, CRS-HIPEC offers acceptable outcomes among those with gastric PCC and long survival for patients without PCC