Secretion systems require high-fidelity mechanisms to discriminate substrates among the vast cytoplasmic pool of proteins. Factors mediating substrate recognition by the type VI secretion system ...(T6SS) of Gram-negative bacteria, a widespread pathway that translocates effector proteins into target bacterial cells, have not been defined. We report that haemolysin coregulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules. Electron microscopy analysis of an Hcp-effector complex from Pseudomonas aeruginosa revealed the effector bound to the inner surface of Hcp. Further studies demonstrated that interaction with the Hcp pore is a general requirement for secretion of diverse effectors encompassing several enzymatic classes. Though previous models depict Hcp as a static conduit, our data indicate it is a chaperone and receptor of substrates. These unique functions of a secreted protein highlight fundamental differences between the export mechanism of T6 and other characterized secretory pathways.
•Hcp is a chaperone required for the intracellular accumulation of Tse2•Export of type VI secretion effectors requires direct interaction with the pore of Hcp•Cognate Hcp-effector interactions can define substrate specificity of the T6SS•Substrate recognition and energization of secretion are uncoupled in the T6SS
Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted ...proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A1 and A2 activity, which are common in host-cell-targeting bacterial toxins and the venoms of certain insects and reptiles. However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors. Our analyses indicate that PldA of Pseudomonas aeruginosa, a eukaryotic-like phospholipase D, is a member of the type VI lipase effector superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). Although previous studies have specifically implicated PldA and the H2-T6SS in pathogenesis, we uncovered a specific role for the effector and its secretory machinery in intra- and interspecies bacterial interactions. Furthermore, we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine, the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the continuing evolution of pathogenesis.
Dental caries can be described as a dysbiosis of the oral microbial community, in which acidogenic, aciduric, and acid-adapted bacterial species promote a pathogenic environment, leading to ...demineralization. Alkali generation by oral microbes, specifically via arginine catabolic pathways, is an essential factor in maintaining plaque pH homeostasis. There is evidence that the use of arginine in dentifrices helps protect against caries. The aim of the current study was to investigate the mechanistic and ecological effect of arginine treatment on the oral microbiome and its regulation of pH dynamics, using an in vitro multispecies oral biofilm model that was previously shown to be highly reflective of the in vivo oral microbiome. Pooled saliva from 6 healthy subjects was used to generate overnight biofilms, reflecting early stages of biofilm maturation. First, we investigated the uptake of arginine by the cells of the biofilm as well as the metabolites generated. We next explored the effect of arginine on pH dynamics by pretreating biofilms with 75 mM arginine, followed by the addition of sucrose (15 mM) after 0, 6, 20, or 48 h. pH was measured at each time point and biofilms were collected for 16S sequencing and targeted arginine quantification, and supernatants were prepared for metabolomic analysis. Treatment with only sucrose led to a sustained pH drop from 7 to 4.5, while biofilms treated with sucrose after 6, 20, or 48 h of preincubation with arginine exhibited a recovery to higher pH. Arginine was detected within the cells of the biofilms, indicating active uptake, and arginine catabolites citrulline, ornithine, and putrescine were detected in supernatants, indicating active metabolism. Sequencing analysis revealed a shift in the microbial community structure in arginine-treated biofilms as well as increased species diversity. Overall, we show that arginine improved pH homeostasis through a remodeling of the oral microbial community.
Sophisticated mechanisms are employed to facilitate information exchange between interfacing bacteria. A type VI secretion system (T6SS) of Pseudomonas aeruginosa was shown to deliver cell ...wall-targeting effectors to neighboring cells. However, the generality of bacteriolytic effectors and, moreover, of antibacterial T6S remained unknown. Using parameters derived from experimentally validated bacterial T6SS effectors we identified a phylogenetically disperse superfamily of T6SS-associated peptidoglycan-degrading effectors. The effectors separate into four families composed of peptidoglycan amidase enzymes of differing specificities. Effectors strictly co-occur with cognate immunity proteins, indicating that self-intoxication is a general property of antibacterial T6SSs and effector delivery by the system exerts a strong selective pressure in nature. The presence of antibacterial effectors in a plethora of organisms, including many that inhabit or infect polymicrobial niches in the human body, suggests that the system could mediate interbacterial interactions of both environmental and clinical significance.
► Mass spectrometry defines several antibacterial T6SS substrates ► T6SS substrates from disparate species reveal an effector-immunity paradigm ► Informatic analysis based on shared properties defines a large T6S effector family ► Effector-immunity distribution indicates a broad role for T6SS in bacterial interactions
Young Indigenous children in North America suffer from a higher degree of severe early childhood caries (S-ECC) than the general population, leading to speculation that the etiology and ...characteristics of the disease may be distinct in this population. To address this knowledge gap, we conducted the first microbiome analysis of an Indigenous population using modern molecular techniques. We investigated the caries-associated microbiome among Canadian First Nations children with S-ECC. Thirty First Nations children <72 mo of age with S-ECC and 20 caries-free children were recruited in Winnipeg, Canada. Parents or caregivers completed a questionnaire on general and dental health, diet, and demographics. The plaque microbiome was investigated by sequencing the 16S rRNA gene. Sequences were clustered into operational taxonomic units and taxonomy assigned via the Human Oral Microbiome Database, then analyzed at the community level with alpha and beta diversity measures. Compared with those who were caries free, children with S-ECC came from households with lower income; they were more likely to live in First Nations communities and were more likely to be bottle-fed; and they were weaned from the bottle at a later age. The microbial communities of the S-ECC and caries-free groups did not differ in terms of species richness or phylogenetic diversity. Beta diversity analysis showed that the samples significantly clustered into groups based on caries status. Twenty-eight species-level operational taxonomic units were significantly different between the groups, including Veillonella HOT 780 and Porphyromonas HOT 284, which were 4.6- and 9-fold higher, respectively, in the S-ECC group, and Streptococcus gordonii and Streptococcus sanguinis, which were 5- and 2-fold higher, respectively, in the caries-free group. Extremely high levels of Streptococcus mutans were detected in the S-ECC group. Overall, First Nations children with S-ECC have a significantly different plaque microbiome than their caries-free counterparts, with the S-ECC group containing higher levels of known cariogenic organisms.
Background
Up to 7.5% of tined-lead removals in patients having sacral neuromodulation (SNM) therapy are associated with a lead breakage. It is still unclear what adverse effects can be caused by ...unretrieved fragments. The aim of our study was to describe the lead removal technique we have been using for the last 2 years in our centre.
Methods
We retrospectively enrolled patients who had lead removal between January 2018 and January 2020 using our standardized technique. The novelty of the technique is in the use of the straight stylet, which is available in the quadripolar tined-lead kit. The stylet gives the electrode greater stiffness, reducing interactions with surrounding tissues and probability of damage or breakage during removal.
Results
In 59 patients (42 women, mean age 57.2 years range 40–79 years) the lead was removed using our standardized technique. In 44 of 59 patients, the tined-lead was removed within 2 months from the SNM-test, due to lack of beneficial effects. In 15 patients the electrode was removed because of failure of definitive implantation. Meantime from definitive implantable pulse generator (IPG) implantation to lead removal was 67.9 months. We recorded only 1 case of lead-breakage during removal: a female patient with a non-tined lead fixed on sacral bone, placed 18 years previously using an open technique.
Conclusions
Lead breakage during removal is not uncommon and adverse effects of retained fragments may occur. Our technique has been safely used for the last 2 years in our centre, with no episodes of lead breakage or retained fragments, except for one non-tined electrode.
Interbacterial interaction pathways play an important role in defining the structure and complexity of bacterial associations. A quantitative description of such pathways offers promise for ...understanding the forces that contribute to community composition. We developed time-lapse fluorescence microscopy methods for quantitation of interbacterial interactions and applied these to the characterization of type VI secretion (T6S) in Pseudomonas aeruginosa . Our analyses allowed a direct determination of the efficiency of recipient cell lysis catalyzed by this intercellular toxin delivery pathway and provided evidence that its arsenal extends beyond known effector proteins. Measurement of T6S apparatus localization revealed correlated activation among neighboring cells, which, taken together with genetic data, implicate the elaboration of a functional T6S apparatus with a marked increase in susceptibility to intoxication. This possibility was supported by the identification of T6S-inactivating mutations in a genome-wide screen for resistance to T6S-mediated intoxication and by time-lapse fluorescence microscopy analyses showing a decreased lysis rate of recipient cells lacking T6S function. Our discoveries highlight the utility of single-cell approaches for measuring interbacterial phenomena and provide a foundation for studying the contribution of a widespread bacterial interaction pathway to community structure.
Xylosandrus germanus (Blandford) has caused increasing damage in high-density New York apple orchards since 2013, resulting in tree decline and death. We documented their occurrence and timing in > ...50 orchards using ethanol-baited traps from 2014 to 2016. First captures ranged from 48 to 83 degree days (base 10 °C) from 1 January. Captures were numerically higher at the orchard–woods interface than within the orchard interior, but differences were not significant in locations with lower populations. Control using insecticide trunk sprays was tested in potted, waterlogged apple trees placed in orchards and nurseries, and inside wooded areas adjacent to orchards. A verbenone repellent was used in combination with trunk sprays to improve control. Overall, insecticide sprays were inconsistent and marginal in preventing new infestations. Chlorpyrifos significantly reduced infestations versus lambda-cyhalothrin and untreated trees at one location in the 2015 orchard trials, and versus untreated trees at one location in the 2016 nursery trials, but otherwise performed no better than other treatments. The addition of verbenone to either the check or permethrin treatments resulted in significantly fewer attack sites containing brood at one orchard site in 2016. Chlorpyrifos, lambda-cyhalothrin, and permethrin significantly reduced the number of attack sites containing adults compared with untreated trees at one nursery trial location in 2016, but were otherwise ineffective in reducing numbers of trees in other locations and infestation categories. We found several fungal and bacterial species associated with X. germanus and its infestation of apples. These microbes likely play a minimal role in apple decline.
The sea urchin embryo is a suitable model that offers an excellent opportunity to investigate different defence strategies activated in stress conditions. We previously showed that cadmium ...accumulates in a dose- and time-dependent manner into embryonic cells, activating different stress and defence mechanisms, including the synthesis of HSPs and the onset of apoptosis and/or autophagy. In this paper we investigated the functional relationship between autophagy and apoptosis, evaluating apoptosis signals in cadmium-exposed Paracentrotus lividus embryos with inhibited autophagy. We found that the inhibition of autophagy produced the concurrent reduction of apoptosis, suggesting that the two phenomena are functionally related. Considering the catabolic role of autophagy, an energetic hypothesis to explain the relationship was evaluated. Using a substrate for ATP production, we found that apoptosis, assessed by TUNEL and cleaved caspase-3 immunocytochemistry, was substantially restored in cadmium-treated embryos where autophagy was inhibited by 3-Methyladenine. On the basis of these results, we propose that, autophagy could play a crucial role in stress response of this organism because autophagy could energetically contribute to apoptotic execution through its catabolic role.
•Investigation on the functional relationship between autophagy and apoptosis.•Evaluation of apoptosis signals in cadmium-exposed Paracentrotus lividus embryos with inhibited autophagy.•How autophagy could energetically contribute to apoptotic execution program, through its catabolic role.
The Multi Effect Distillation (MED) process is often proposed as a key technology for the construction of new thermal desalination plants, especially within solar-powered cogeneration schemes. With ...this respect, the need for transient behaviour analysis requires the development of dynamic models for the MED process. Only a few have been presented so far in the literature, in which, however, several simplifying assumptions and constrains are still limiting their potential use.
The model here proposed addresses most of the aspects still limiting previous models formulations. The powerful equation-based process simulator gPROMS® was chosen for the implementation of the model, developed on the basis of available data from a MED-TVC plant located in Trapani, Sicily (Italy). After validation, the model was used for some preliminary analysis of system behaviour under transient conditions, artificially generated starting from steady state by implementing specific disturbances such as the variation in the motive steam pressure, seawater feed flow rate and temperature. This demonstrates the model capability to describe in detail the dynamic response of the system with respect to all its variables, thus representing a useful tool for the prediction of transient operations and control system design purposes of MED-TVC plants.
•An equation-based dynamic model for MED-TVC has been implemented.•It also accounts for non-condensables, partial condensation & brine level variation.•Transient operations due to disturbances are analysed.•Critical transitory conditions have been identified.•Strategies for process control under variable regime are discussed.
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