High tolerance to mutations in a Chlamydia trachomatis peptide deformylase loop

AIM: To determine if and how a loop region in the peptide deformylase (PDF) of Chlamydia trachomatis regulates enzyme function.METHODS: Molecular dynamics simulation was used to study a structural model of the chlamydial PDF (cPDF) and predict the temperature factor per residue for the protein backbone atoms. Site-directed mutagenesis was performed to construct cPDF variants. Catalytic properties of the resulting variants were determined by an enzyme assay using formyl-Met-Ala-Ser as a substrate.RESULTS: In silico analysis predicted a significant increase in atomic motion in the DGELV sequence (residues 68-72) of a loop region in a cPDF mutant, which is resistant to PDF inhibitors due to two amino acid substitutions near the active site, as compared to wild-type cPDF. The D68R and D68R/E70R cPDF variants demonstrated significantly increased catalytic efficiency. The E70R mutant showed only slightly decreased efficiency. Although deletion of residues 68-72 resulted in a nearly threefold loss in substrate binding, this deficiency was compensated for by increased catalytic efficiency.CONCLUSION: Movement of the DGELV loop region is involved in a rate-limiting conformational change of the enzyme during catalysis. However, there is no stringent sequence requirement for this region for cPDF enzyme activity.     

2017: beginning of a new era for Chlamydia research in China and the rest of the world

The First Chinese Chlamydia Research Meeting was held in Lanzhou, China in May 2017, 60 years after the disclosure of reproducible isolation of Chlamydia trachomatis by 汤飞凡 (Fei-fan Tang). We report current state of the Chlamydia research community in China, and briefly review recent progress in Chlamydia vaccinology. The meeting represents a new milestone for Chlamydia research in the country. The Chinese Chlamydia Research Society (CCRS) was formed during the meeting. Future meetings will be held biennially and should facilitate collaboration of Chinese researchers with their domestic and international colleagues.     

Localization of TLR2 and MyD88 to Chlamydia trachomatis inclusions. Evidence for signaling by intracellular TLR2 during infection with an obligate intracellular pathogen

Chlamydia trachomatis is an obligate intracellular gram-negative pathogen and the etiologic agent of significant ocular and genital tract diseases. Chlamydiae primarily infect epithelial cells, and the inflammatory response of these cells to the infection directs both the innate and adaptive immune response. This study focused on determining the cellular immune receptors involved in the early events following infection with the L2 serovar of C. trachomatis.We found that dominant negative MyD88 inhibited interleukin-8 (IL-8) secretion during a productive infection with chlamydia. Furthermore, expression of Toll-like receptor (TLR)-2 was required for IL-8 secretion from infected cells, whereas the effect of TLR4/MD-2 expression was minimal. Cell activation was dependent on infection with live, replicating bacteria, because infection with UV-irradiated bacteria and treatment of infected cells with chloramphenicol, but not ampicillin, abrogated the induction of IL-8 secretion. Finally, we show that both TLR2 and MyD88 co-localize with the intracellular chlamydial inclusion, suggesting that TLR2 is actively engaged in signaling from this intracellular location. These data support the role of TLR2 in the host response to infection with C. trachomatis. Our data further demonstrate that TLR2 and the adaptor MyD88 are specifically recruited to the bacterial or inclusion membrane during a productive infection with chlamydia and provide the first evidence that intracellular TLR2 is responsible for signal transduction during infection with an intracellular bacterium.     

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.     

Molecular cloning of a sulfotransferase in Arabidopsis thaliana and regulation during development and in response to infection with pathogenic bacteria

A cDNA clone (RaRO47) encoding a sulfotransferase (ST) has been isolated from Arabidopsis cell suspensions. The deduced polypeptide of 302 amino acids is highly related to plant flavonol sulfotrans-ferases (FSTs), characterized for the first time in Flaveria, and also to STs from animal tissue. The expression of the Arabidopsis ST gene(s) corresponding to RaR047 was examined during different developmental stages. It was found that, at the level of steady-state mRNA, expression of gene(s) encoding this ST was rapidly induced in the aerial parts of young seedlings, and during growth of Arabidopsis cell cultures. No expression could be detected in roots. Treatment of Arabidopsis seedlings with hormonal or stress-related compounds, showed that RaR047 mRNA accumulation was more particularly induced in response to salicylic acid and methyl jasmonate. Furthermore, in the leaves of mature plants or in cell suspensions, accumulation of RaR047 mRNA was observed upon infection with bacterial pathogens. This expression was observed preferentially in response to avirulent pathogens causing an hyper-sensitive reaction, as compared to virulent pathogens, which lead to disease.     

Zinc levels of patients with a moderate to severe COVID-19 infection at hospital admission and after 4th days of ward hospitalization and their clinical outcome

BackgroundPrevious studies associate the disturbance of the Zinc (Zn) status with the severity of the disease and the inflammatory process in the critically ill patient. This decrease in Zn concentrations is an indicator of poor prognosis. Our aim was to evaluate Zn levels at admission and after four days, and to study if lower Zn levels at those days were related to a worse clinical outcome.Material and methodsObservational cohort study at a tertiary Hospital. Recruitment period: 09/04/2020–04/24/2021. Clinical information on hypertension, diabetes mellitus (DM), chronic obstructive pulmonary disease (COPD), or bronchial asthma was collected. Obesity was defined as BMI ≥ 30 Kg/m2. Blood extraction was performed at admission and after 4 days. Zn was measured by atomic absorption using a flame method. Worse clinical outcome was defined as death during admission, intensive critical care unit admission or receiving supplemental oxygen through noninvasive or invasive ventilator care.Results129 subjects were invited to participate but only 100 subjects completed the survey. According to ROC curve [AUC= 0.63 (95% CI 0.60–0.66)], Zn < 79 μg/dL showed the best performance to detect a worse outcome (Sn=0.85; Sp=0.36). Patients with Zn < 79 μg/dL were older (70 vs 61 y; p = 0.002) with no differences by sex. Most patients presented with fever, dysthermic symptoms and cough, without differences between groups. Pre-existing comorbid conditions did not differ significantly between groups. Less obese subjects were found in the Zn < 79 μg/dL group (21.4 vs 43.3%, p = 0.025). In the univariate analysis, Zn < 79 μg/dL at hospital admission was related to a worse outcome (p = 0.044), but after adjusting for age, C-reactive protein, and obesity there was no difference, but a tendency towards a worse prognosis [OR 2.20 (0.63–7.70), p = 0.215]. Zn levels increased in both groups after 4 days (66.6 vs 73.1 μg/dL at admission, and 72.2 vs 80.5 μg/dL at 4th day), with ns. difference (p = 0.214).ConclusionZn < 79 μg/dL at admission for a moderate to severe COVID-19 infection could be related to a worse outcome, although after adjustment for age, C-reactive protein levels and obesity, this Zn level threshold did not show statistically significant difference in the composite end point, but a tendency towards a worse prognosis. In addition, patients with the best clinical evolution showed higher serum Zn levels at 4th day after hospital admission than the patients with a worse prognosis.