Biology subjects

Gangi Setty, S. R.

Publications and source records attributed to Gangi Setty, S. R..

7 recordsLinked to original sources

bioRxiv2024

Salmonella Typhimurium Manipulates Syntaxin 7 to Navigate Endo-Lysosomal Trafficking in Host Cells

Intracellular pathogens rely on manipulating host endocytic pathways to ensure survival. Legionella and Chlamydia exploit host SNARE proteins, with Legionella cleaving syntaxin 17 (STX17) and Chlamydia interacting with VAMP8 and VAMP7. Similarly, Salmonella targets the hosts endosomal fusion machinery, using SPI effectors like SipC and SipA to interact with syntaxin 6 (STX6) and syntaxin 8 (STX8), respectively, maintaining its vacuolar niche. Recent evidence highlights syntaxin 7 (STX7), a Qa-SNARE involved in endo-lysosomal fusion, as a potential Salmonella target. BioID screening revealed STX7 interactions with SPI-2 effectors SifA and SopD2, suggesting a critical role in Salmonella pathogenesis. We investigated the role of STX7 in Salmonella-containing vacuole (SCV) biogenesis and pathogenesis in macrophages and epithelial cells. Our findings indicate that STX7 levels and localization differ between these cell types during infection, reflecting the distinct survival strategies of Salmonella. Live cell imaging showed that STX7 is recruited to SCVs at different infection stages, with significantly altered distribution in HeLa cells at the late stage of infection. STX7 knockdown resulted in reduced bacterial survival, which was rescued upon overexpression of STX7 in both HeLa and RAW264.7 cells, suggesting Salmonella hijacks STX7 to evade lysosomal fusion and secure nutrients for intracellular replication. These results underscore the essential role of STX7 in maintaining SCVs and facilitating Salmonella survival. Further, the temporal expression of STX7 adaptor/binding partners in macrophages showed dynamic interactions with STX7 facilitating Salmonella infection and survival in host cells. Together, our study highlights STX7 as a critical host factor exploited by Salmonella, providing insights into the molecular mechanisms underlying its pathogenesis in macrophages and epithelial cells. These findings may in form strategies for targeting host-pathogen interactions to combat Salmonella infections.

microbiology↗

bioRxiv2024

ER stress and mitochondrial dynamics: a tale of a wandering phosphatase DUSP28

Fission and fusion processes maintain the mitochondrial dynamics at a steady state and are altered during various cellular stresses. The mechanisms that regulate mitochondrial dynamics during ER stress are critically unknown. Here, we identified a dual specificity phosphatase DUSP28 with a novel role in regulating mitochondrial morphology during ER stress. Cytosolic DUSP28 translocates to mitochondria during ER stress via PERK activation and further promotes mitochondrial fission in a Drp1-dependent manner. Interestingly, the knockdown of DUSP28 activates PERK signaling and enhances ER expansion following mitochondrial elongation by enhancing S637 phosphorylation of Drp1. Overexpression of DUSP28-GFP could not rescue the loss of Drp1 function, indicating its necessity for mitochondrial fission. Further, the presence of DUSP28 on mitochondria prevents Parkin recruitment during CCCP-induced mitochondrial damage and follows STX17-dependent mitophagy. These findings illustrate the novel crosstalk between ER stress and DUSP28 phosphatase in maintaining the mitochondrial dynamics to sustain cellular stress. Key pointsO_LIPERK activation translocates cytosolic DUSP28 to mitochondria C_LIO_LIDUSP28 mediates Drp1-dependent mitochondrial fission C_LIO_LIDUSP28 phosphorylation status determines its localization and function C_LIO_LIDUSP28 inhibits Parkin recruitment to mitochondria upon CCCP treatment and follows STX17-mediated mitophagy C_LI

cell biology↗

bioRxiv2024

Endoplasmic Reticulum contact sites facilitate the coordinated division of Salmonella-containing vacuole (SCV)

Salmonella Typhimurium (STM) resides in a membrane-bound compartment called Salmonella containing vacuole (SCV) in several infected cell types. Within host cells, the division of bacteria and SCV are synchronous to maintain the single bacterium per vacuole. However, the mechanism regulating the synchronous fission and the machinery is not well understood. The fission of several intracellular organelles is regulated by the dynamic nature of the tubular endoplasmic reticulum (ER). In this study, we have evaluated the role of ER in controlling SCV fission. Interestingly, Salmonella-infected cells show the activation of unfolded protein response (UPR) with expanded ER tubules compared to the uninfected cells. Further, changing the expression of ER morphology regulators, such as reticulon-4a (Rtn4a) and CLIMP63, affected bacterial proliferation significantly, suggesting a potential role for tubular ER in facilitating the SCV division. Live-cell imaging analysis shows the marking of tubular ER precisely at the center of the majority of SCV division (78%) sites. We have investigated the role of SteA (a known Salmonella effector in modulating the membrane dynamics) in coordinating the SCV division. We observed that SteA resides on the SCV membranes and helps in making membrane contact sites between SCV and ER. Accordingly, the colocalization of ER with SCV enclosing SteA mutant Salmonella was significantly reduced compared to SCV-formed by wild-type Salmonella. Depletion of steA in Salmonella resulted in profound defects in SCV division, resulting in multiple bacteria residing in a single vacuole with defects in proliferation compared to the wild-type strain in epithelial cells. Also, during in vivo infection, the STM{Delta}steA mutant shows a defect in colonization in the spleen and liver and affects the initial survival rate of mice. Overall, this study suggests a coordinated role of bacterial effector SteA in promoting the ER contact sites with SCVs and thus regulating the successful division of SCV. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/592158v2_ufig1.gif" ALT="Figure 1000"> View larger version (72K): org.highwire.dtl.DTLVardef@21e83aorg.highwire.dtl.DTLVardef@157052org.highwire.dtl.DTLVardef@1815308org.highwire.dtl.DTLVardef@1724888_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2022

Golgi apposition promotes the generation of specialized lysosomes in differentiated keratinocytes

Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling in lysosomes and the Golgi apparatus. However, the functional significance and the molecular link between lysosome and Golgi remodeling were unknown. Here, we show that in differentiated keratinocytes, the Golgi apparatus redistributes as ministacks leading to a significant increase in total protein secretion. The Golgi ministacks establish contact with lysosomes facilitated by Golgi tethering protein GRASP65, the depletion of which is associated with the loss of Golgi-lysosome contact and malformation of lysosomes defined by their aberrant morphology, size, and function. Strikingly, these lysosomes receive secretory Golgi cargoes, contribute to the protein secretion from the Golgi, and are critically maintained by the secretory function of the Golgi apparatus. We uncovered a novel mechanism of lysosome specialization through unique Golgi-lysosome contact that likely supports high secretion from differentiated keratinocytes. Key pointsO_LICalcium induced differentiation of human keratinocytes accompanies the dispersal of functional Golgi stacks and lysosomes. C_LIO_LIDispersed Golgi stacks establish contact/physical apposition with the lysosomes. C_LIO_LIGolgi tether GRASP65 surrounds keratinocyte lysosomes and facilitates Golgi-lysosome apposition. C_LIO_LIGRASP65 depletion abolishes Golgi-lysosome apposition and accumulates morphologically altered non-degradative lysosomes. C_LIO_LILysosomes of differentiated keratinocytes receive secretory Golgi cargo and contribute to the protein secretion from the Golgi. C_LIO_LISpecialized lysosomes are maintained by the secretory function of the Golgi apparatus. C_LI

cell biology↗

bioRxiv2022

Syntaxin 3-SPI 2 dependent cross-talk facilitates the division of Salmonella containing vacuole (SCV)

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in the vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. A report showed that Salmonella containing vacuole (SCV) harbors recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis is unclear. Upon knockdown of STX3, we have observed a reduction in bacterial proliferation and is restored upon the overexpression of STX3. Post infected live-cell imaging of cells showed STX3 localises to the SCV membranes and thus might help in fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found this interaction abrogated when we infected with SPI-2 encoded T3SS apparatus mutant (STM {Delta}ssaV) but not with SPI-1 encoded T3SS (STM{Delta} invC). These observations were also consistent in mice model of Salmonella infection. Together, these results shed a light on the effector molecules secreted through SPI-2 encoded by T3SS possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and maintenance the principle single bacterium per vacuole. SynopsisSalmonella Typhimurium infection in murine macrophage leads to upregulation of host Syntaxin 3 both at transcript and protein levels at late stage of infection. Syntaxin 3 cross-talk with Salmonella containing vacuoles (SCVs) is essential for establishment of replicative niche in host macrophages. The cross-talk between STX3 and SCVs is Salmonella pathogenicity island 2 (SPI-2) dependent and is consistent in mice model of Salmonella Typhimurium infection.

microbiology↗

bioRxiv2022

Deceiving The Big Eaters: Salmonella Typhimurium SopB subverts host cell Xenophagy through the Akt-TFEB axis in macrophages

Salmonella, a stealthy facultative intracellular pathogen, harbors an array of host immune evasion strategies. This facilitates successful survival and replicative niches establishment in otherwise hostile host innate immune cells such as macrophages. Salmonella survives and utilizes macrophages for effective dissemination throughout the host causing systemic infection. One of the central host defense mechanisms in macrophages is bacterial xenophagy or macro-autophagy. Here we report for the first time that Salmonella pathogenicity island-1 (SPI-1) effector SopB is involved in subverting host autophagy through dual mechanisms. SopB is known to act as a phosphoinositide phosphatase and thereby can alter the phosphoinositide dynamics of the host cell. Here we demonstrate that this activity helps the bacterium escape autophagy by inhibiting terminal fusion of Salmonella containing vacuole (SCV) with both lysosomes and autophagosomes. We also report the second mechanism, wherein SopB downregulates overall lysosomal biogenesis through Akt- transcription factor EB (TFEB) axis. TFEB is a master regulator of lysosomal biogenesis and autophagy, and SopB restricts the nuclear localization of TFEB. This reduces the overall lysosome content inside host macrophages, further facilitating survival in macrophages and systemic dissemination of Salmonella in the host.

microbiology↗

bioRxiv2018

A non-canonical lysosome biogenesis pathway generates Golgi-associated lysosomes during epidermal differentiation

Keratinocytes maintain epidermis integrity and function including physical and antimicrobial barrier through cellular differentiation. This process is predicted to be controlled by calcium ion gradient and nutritional stress. Keratinocytes undergo proteome changes during differentiation, which enhances the intracellular organelle digestion to sustain the stress conditions. However, the molecular mechanism between epidermal differentiation and organelle homeostasis is poorly understood. Here, we used primary neonatal human epidermal keratinocytes to study the link between cellular differentiation, signaling pathways and organelle turnover. Upon addition of calcium chloride (2 mM) to the culture medium, keratinocytes increased their cell size and the expression of differentiation markers. Moreover, differentiated keratinocytes showed enhanced lysosome biogenesis that was dependent on ATF6-arm of UPR signaling but independent of mTOR-MiT/TFE transcription factors. Furthermore, chemical inhibition of mTOR has increased keratinocyte differentiation and relocalized the MiT/TFE TFs to the lysosome membranes, indicating that autophagy activation promotes the epidermal differentiation. Interestingly, differentiation of keratinocytes resulted in dispersal of fragmented Golgi and lysosomes, and the later organelles showed colocalization with Golgi-tethering proteins, suggesting that these lysosomes possibly originated from Golgi, hence named as Golgi-associated lysosomes (GALs). Consistent to this prediction, inhibition of Golgi function using brefeldin A completely abolished the formation of GALs and the keratinocyte differentiation. Thus, ER stress regulates the biogenesis of GALs, which maintains keratinocyte differentiation and epidermal homeostasis.

cell biology↗