Pyruvate oxidase as a key determinant of pneumococcal viability during transcytosis across brain endothelium.

Streptococcus pneumoniae (SPN/pneumococcus), invades myriad of host tissues following efficient breaching of cellular barriers. However, strategies adopted by pneumococcus for evasion of host intracellular defences governing successful transcytosis across host cellular barriers remain elusive. In this study, using brain endothelium as a model host barrier, we observed that pneumococcus containing endocytic vacuoles (PCVs) formed following SPN internalization into brain microvascular endothelial cells (BMECs), undergo early maturation and acidification, with a major subset acquiring lysosome-like characteristics. Exploration of measures that would preserve pneumococcal viability in the lethal acidic pH of these lysosome-like vacuoles revealed a critical role of the two-component system response regulator, CiaR, which has been previously implicated in induction of acid tolerance response. Pyruvate oxidase (SpxB), a key sugar metabolizing enzyme that catalyses oxidative decarboxylation of pyruvate to acetyl phosphate, was found to contribute to acid stress tolerance, presumably via acetyl phosphate-mediated phosphorylation and activation of CiaR, independent of its cognate kinase CiaH. Hydrogen peroxide, the by-product of SpxB catalysed reaction, was also found to improve pneumococcal intracellular survival by oxidative inactivation of lysosomal cysteine cathepsins, thus compromising the degradative capacity of the host lysosomes. Expectedly, a Δ spxB mutant was found to be significantly attenuated in its ability to survive inside the BMEC endocytic vacuoles, reflecting in its reduced transcytosis ability. Collectively, our studies establish SpxB as an important virulence determinant facilitating pneumococcal survival inside host cells, ensuring successful trafficking across host cellular barriers. IMPORTANCE Host cellular barriers have innate immune defences to restrict microbial passage into sterile compartments. Here, by focussing on the blood-brain barrier endothelium, we investigated mechanisms which enable Streptococcus pneumoniae to traverse through host barriers. Pyruvate oxidase, a pneumococcal sugar metabolizing enzyme was found to play a crucial role in this, via generation of acetyl phosphate and hydrogen peroxide. A two-pronged approach consisting of acetyl phosphate-mediated activation of acid tolerance response and hydrogen peroxide-mediated inactivation of lysosomal enzymes enabled pneumococci to maintain viability inside the degradative vacuoles of the brain endothelium, for successful transcytosis across the barrier. Thus, pyruvate oxidase is a key virulence determinant and can potentially serve as a viable candidate for therapeutic interventions for better management of invasive pneumococcal diseases.

The acid tolerance response and pH adaptation of Enterococcus faecalis in extract of lime Citrus aurantiifolia from Aceh Indonesia

Background: The objective of the present study was to evaluate the acid tolerance response and pH adaptation whenEnterococcusfaecalisinteracted with extract of lime (Citrus aurantiifolia).Methods:We usedE. faecalisATCC 29212 and lime extract from Aceh, Indonesia. The microbe was analyzed for its pH adaptation, acid tolerance response, and adhesion assay using a light microscope with a magnification of x1000. Further, statistical tests were performed to analyze both correlation and significance of the acid tolerance and pH adaptation as well as the interaction activity.Results:E. faecaliswas able to adapt to a very acidic environment (pH 2.9), which was characterized by an increase in its pH (reaching 4.2) at all concentrations of the lime extract (p < 0.05).E. faecaliswas also able to provide acid tolerance response to lime extract based on spectrophotometric data (595 nm) (p < 0.05). Also, the interaction activity ofE. faecalisin different concentrations of lime extract was relatively stable within 6 up to 12 hours (p < 0.05), but it became unstable within 24–72 hours (p > 0.05) based on the mass profiles of its interaction activity.Conclusions:E. faecaliscan adapt to acidic environments (pH 2.9–4.2); it is also able to tolerate acid generated byCitrus aurantiifoliaextract, revealing a stable interaction in the first 6–12 hours.