Biology subjects

Chandrasekharan, G.

Publications and source records attributed to Chandrasekharan, G..

3 recordsLinked to original sources

bioRxiv2025

The staphylococcal type VII secretion system delays macrophage cell death through modulating multiple cell death pathways

The major human pathogen Staphylococcus aureus is a facultatively intracellular bacterium that can replicate and survive within a range of host cells including macrophages. While macrophage-mediated immune modulation is pivotal in S. aureus pathogenesis, bacterial manipulation of macrophage pathways remains poorly understood. The specialised type VII secretion system (T7SSb) is an important virulence-associated factor and immune modulator during staphylococcal infection, although, unlike its mycobacterial counterparts, its role in controlling staphylococcal-macrophage interactions remains unclear. Employing high-resolution time-lapse imaging, we demonstrated an induction of cell death corpses or pore-induced cellular traps (PITs) during S. aureus infection of macrophages in vitro, with a higher number of PITs forming in macrophages infected with S. aureus lacking EssC ({Delta}essC), a central T7SS transporter. Interestingly, {Delta}essC-infected macrophages displayed increased bacterial escape compared to wild type (WT)-infected cells, implicating a role for T7SS in delaying macrophage cell death. {Delta}essC-infected macrophages were also efferocytosed less compared to WT in vitro. We investigated the induction of cell death signalling markers in WT and {Delta}essC-infected macrophages and demonstrated that the T7SS delayed the induction of key cell death pathways, necroptosis and pyroptosis. A dual RNAseq analysis of WT- and {Delta}essC-infected macrophages further indicated a T7SS-dependent increase in ferroptosis later in infection, along with modulation of chemokines. We demonstrated that individual T7SS effectors, EsxA and EsxC, which were induced within macrophages, could individually delay macrophage cell death. Furthermore, in a murine skin infection model, we showed that effector and transporter mutants have distinct S. aureus infection outcomes. Our findings suggest for the first time a key role for T7SSb proteins in controlling macrophage cell death, which impacts staphylococcal survival within the host during infection. ImportanceStaphylococcus aureus is a major hospital and community associated pathogen which causes an array of infections. S. aureus can survive within immune cells, which may provide a niche for this pathogen to both persist and disseminate. Here we report a role for the staphylococcal type VII secretion system b, which secretes effectors that have been associated with bacterial virulence, in delaying macrophage cell death induced in response to this pathogen. T7SS functions through interfering with inflammatory cell death pathways, impacting the local cellular and immune environment. Our findings thus highlight a protective role for this system in macrophages, distinct to functions reported for the mycobacterial T7SS, and may implicate similar functions for T7SS proteins from related pathogens. Controlling macrophage cell death could be a critical mechanism by which S. aureus modulates the local immune responses, enabling its survival within the host,

microbiology↗

bioRxiv2025

The cellular mammalian clock regulates Staphylococcus aureus invasion in epithelial cells

An endogenous biological clock, the circadian clock, coordinates life with the 24-hour day/night environmental cycle. In mammals, a central pacemaker in the suprachiasmatic nucleus of the hypothalamus coordinates timing between peripheral clocks and with the environment and, for example, modulates immune responses to infections. However, its role in controlling bacterial infections at a cellular level is not understood. Here, we investigate the role of the host cellular clock during infection by a highly drug-resistant human pathogen, Staphylococcus aureus. Our findings revealed that S. aureus invasion into epithelial cells was dependent on the host circadian phase. Interestingly, cells deficient in BMAL1, a transcriptional activator and an essential clock protein, demonstrated increased bacterial uptake compared to parental A549 cells. The BMAL1-knockdown (KD) cells showed significant induction of GP340, a receptor of the S. aureus adhesin, SraP. An S. aureus sraP mutant did not exhibit rhythmic uptake into A549 cells or an increased uptake into BMAL1-KD compared to parental A549 cells. Of note, other bacterial adhesin mutants showed rhythmic and higher uptake in BMAL1-KD cells. Hence, we report that S. aureus epithelial cell invasion is clock-modulated and mediated through the S. aureus SraP-GP340 pathway, suggesting potential for host clock-directed therapy against this pathogen.

microbiology↗

bioRxiv2023

The staphylococcal type VII secretion system impacts daptomycin sensitivity through controlling bacterial cell envelope integrity

The human pathogen Staphylococcus aureus encodes a specialised type VII secretion system (T7SS), which plays an important role in bacterial virulence during infection. However, the functions the T7SS during infection and in bacterial physiology remain unclear. Here we demonstrate that S. aureus strains lacking the the T7SS effector EsxC ({Delta}esxC) was highly sensitive to the important last resort drug, daptomycin, as well as other membrane-targeting antibiotics, including gramicidin and bithionol. To understand how EsxC mediates increased antibiotic sensitivity, we investigated its functions in the staphylococcal cell envelope. Scanning electron microscopy analysis of an esxC mutant revealed a distinct cell surface morphology. Interestingly, {Delta}esxC displayed a decrease in membrane fluidity, altered membrane protein profiles and altered cell wall synthesis. The esxC mutant demonstrated enhanced daptomycin binding which correlated with the increased negative charge of mutant membranes. Calcium ions, which can bind membranes affecting charge, impacted growth of {Delta}esxC and sensitivity to daptomycin, suggesting that EsxC may modulate calcium binding to membranes. Furthermore, the esxC mutant displayed a heightened susceptibility to daptomycin during intracellular infection, and in a murine skin infection model. Thus, our data show that the T7SS effector EsxC impacts sensitivity of S. aureus to membrane-acting drugs such as daptomycin through modulation of cell membrane integrity, indicating its potential as a drug target. Author SummaryT7SS has a range of functions in bacteria including specific roles in bacterial physiology including DNA uptake, membrane integrity and bacterial development. In S. aureus T7SS has been shown to be critical for bacterial virulence, intra-species competition and in host cell interactions, although their functions in bacterial physiology are not clear. Here we report a role of the staphylococcal T7SS effector EsxC in the modulation of the cell membrane and surface integrity, which impacts the activity of membrane targeting drugs like daptomycin. Our data indicate that targeting this system could potentially enhance activity of existing therapeutic agents.

microbiology↗