Biology subjects

Bobrovskyy, M.

Publications and source records attributed to Bobrovskyy, M..

3 recordsLinked to original sources

bioRxiv2025

Type VIIb secretion system recruits a dedicated cell wall hydrolase EssH to enable effector secretion by Staphylococcus aureus

Staphylococcus aureus is a pervasive human pathogen that heavily relies on protein secretion to exert its virulence strategies. S. aureus encodes a specialized type VIIb secretion system (T7SSb) that contributes to virulence and persistence in the host. T7SSb supports secretion of small proteins belonging to a WXG100 family as well as larger LXG polymorphic toxins. Secretion of these proteins is facilitated by the T7SSb complex that assembles in the cell envelope from core components EsaA, EssA, EssB, and the conserved ATPase EssC. T7SSb-mediated secretion also requires the cell wall hydrolase EssH that bears a cystine histidine-dependent amidase/peptidase (CHAP) domain. Hereby, we show that the N-terminal domain of EssH functions together with the CHAP domain to support T7SSb secretion. We find that EssH copurifies with the T7SSb core components and is required for the assembly of EsaA across the cell wall. Finally, secreted EssH is released into the extracellular milieu and degraded by the secreted staphylococcal protease, staphopain A. We propose a model to capture the function of the dedicated cell wall hydrolase EssH for T7SSb in S. aureus. IMPORTANCEStaphylococcus aureus is a leading cause of infections worldwide. S. aureus utilizes a specialized type VIIb secretion system (T7SSb) to persist in the infected host tissues as well as target competitor bacteria to establish its niche. T7SSb assembles into a multiprotein translocation complex and facilitates secretion of a set of small proteins and larger polymorphic toxins across the cytosolic membrane. Beyond the membrane, secreted proteins were thought to diffuse through the thick yet porous cell wall and release into the environment. Here, we demonstrate for the first time that S. aureus T7SSb extends across the cell wall via its EsaA subunit. Furthermore, accommodation of EsaA within the cell wall requires an associated cell wall hydrolase EssH and is essential for protein secretion via T7SSb. Thus, our findings provide a mechanistic insight for a coordinated cell wall processing and T7SSb assembly to support specialized protein secretion in S. aureus.

microbiology↗

bioRxiv2020

Effects of individual base-pairs on in vivo target search and destruction kinetics of small RNA

Base-pairing interactions mediate intermolecular target recognition in many biological systems and applications, including DNA repair, CRISPR, microRNA, small RNA (sRNA) and antisense oligo therapies. Even a single base-pair mismatch can cause a substantial difference in biological activity but presently we do not yet know how the target search kinetics in vivo are influenced by single nucleotide level changes. Here, we used high-throughput sequencing to identify functionally relevant single point mutants of the bacterial sRNA, SgrS, and quantitative super-resolution microscopy to probe the mutational impact on the regulation of its primary target, ptsG mRNA. Our super-resolution imaging and analysis platform allowed us to further dissect mutational effects on SgrS lifetimes, and even subtle changes in the in vivo rates of target association, kon, and dissociation, koff. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decreased kon and increased koff, providing an in vivo demonstration that Hfq directly facilitates sRNA-mRNA annealing. Single base-pair mismatches in the annealing region reduced kon by 24-31% and increased koff by 14-25%, extending the time it takes to find and destroy the target mRNA by about a third, depending on whether an AU or GC base-pair is disrupted. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq also buffers base-pair disruptions.

molecular biology↗

bioRxiv2017

Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS

The mechanisms by which small RNA (sRNA) regulators select and prioritize target mRNAs remain poorly understood, but serve to promote efficient responses to environmental cues and stresses. We sought to uncover mechanisms that establish regulatory hierarchy for a model sRNA, SgrS, found in enteric bacteria and produced under conditions of metabolic stress when sugar transport and metabolism are unbalanced. SgrS post-transcriptionally controls a nine-gene regulon to restore growth and homeostasis under stress conditions. An in vivo reporter system was used to quantify SgrS-dependent regulation of target genes and established that SgrS exhibits a clear preference for certain targets, and regulates those targets efficiently even at low SgrS levels. Higher SgrS concentrations are required to regulate other targets. The position of targets in the regulatory hierarchy is not well-correlated with the predicted thermodynamic stability of SgrS-mRNA interactions or the SgrS-mRNA binding affinity as measured in vitro. Detailed analyses of SgrS interaction with asd mRNA demonstrate that SgrS binds cooperatively to two sites and remodels asd mRNA secondary structure. SgrS binding at both sites increases the efficiency of asd mRNA regulation compared to mutants that have only a single SgrS binding site. Our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features. The sRNA-mRNA interaction, including the number and position of sRNA binding sites on the mRNA and cofactors like the RNA chaperone Hfq, seem to tune the efficiency of regulation of specific mRNA targets.\n\nIMPORTANCETo survive, bacteria must respond rapidly to stress and simultaneously maintain metabolic homeostasis. The small RNA (sRNA) SgrS mediates the response to stress arising from imbalanced sugar transport and metabolism. To coordinate the stress response, SgrS regulates genes involved in sugar uptake and metabolism. Intrinsic properties of sRNAs such as SgrS allow them to regulate extensive networks of genes. To date, sRNA regulation of targets has largely been studied in the context of \"one sRNA-one target\", and little is known about coordination of multi-gene regulons and sRNA regulatory network structure. Here, we explore the molecular basis for regulatory hierarchy in sRNA regulons. Our results reveal a complex interplay of factors that influence the outcome of sRNA regulation. The number and location of sRNA binding sites on mRNA targets and the participation of an RNA chaperone dictate prioritized regulation of targets to promote an efficient response to stress.

microbiology↗