Biology subjects

Chakravortty, D.

Publications and source records attributed to Chakravortty, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Maltose aids in the growth of Salmonella Typhimurium in nitrogen-deficient conditions

Enteric pathogens, such as Salmonella, encounter uncertain nutrients in both the host and the environment. Nutrients such as carbon and nitrogen are essential for growth, and when these nutrients are limited, bacteria have developed various mechanisms to overcome stress and thrive in nutrient-rich environments. In this study, we investigated the role of the disaccharide sugar maltose in the growth and pathogenicity of Salmonella Typhimurium (STM) under nitrogen-deficient culture conditions. Throughout the study, we used Burks medium to mimic nitrogen-deficient conditions. STM growth in Burks medium was minimal, and maltose supplementation enhanced STM growth in both liquid and solid Burks media. The STM grown in Burks medium had a reduced bacterial cell length, which increased upon the addition of maltose and became comparable to that of LB-grown STM WT. Growth was dependent on the NtrBC two-component system and sigma factor RpoE. Deletion of ntrB and rpoE significantly reduced the growth in Burks medium during the log phase. Salmonella grown in Burks medium with maltose had reduced invasion in Caco-2 cells, phagocytosis in RAW 264.7 macrophages, and organ burden in C57BL/6 mice. Our findings revealed the role of maltose in enhancing the growth and length of Salmonella under nitrogen-deficient conditions. However, Salmonella grown in Burks medium with maltose is defective in pathogenesis.

microbiology↗

bioRxiv2026

Eco-friendly approach to indenodiazepinones from o-formylynones and o-phenylenediamines: Novel pharmacophoric constructs and their anti-bacterial activity

We have conceptualised and executed an efficient, eco-friendly, one-flask, synthetic approach of general applicability to functionally enriched and potentially pharmacophoric tetracyclic indenodiazepinones through a base-mediated reaction cascade between a multifunctional o-formylynones and commercial o-phenylenediamines. The pertinence of this operationally simple, no-waste protocol, embodying many green and sustainable features, has been scoped with 26 variegated examples. This controlled one-pot assembly of novel tetracyclic indenodiazepinones with an unusual placement of an olefin through a reaction cascade involving Schiff base formation, intramolecular aza-Michael addition and a Mannich reaction between o-formylynones and o-phenylenediamines rapidly generates a diverse library poised for exploring their therapeutic potential and medicinal chemistry applications. The newly accessed indenodiazepinones exhibit promising antibacterial activities against many microorganisms and are non-cytotoxic even at higher concentrations. Interestingly, one of the compounds, 3v did not develop resistance in Salmonella Typhimurium (STM) upon repeated exposure to sub-lethal doses up to the 160th cycle, whereas resistance against clinically used antibacterial ciprofloxacin was attained at the 128th cycle. These preliminary findings open avenues towards the development of novel antimicrobial agents with enhanced durability against drug resistance and these efforts are disclosed in this report. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/697289v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d1d9beorg.highwire.dtl.DTLVardef@2e8560org.highwire.dtl.DTLVardef@36742eorg.highwire.dtl.DTLVardef@105ae7c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Efficient elimination of microbial infections in wounds using plasma activated water hydrogel

Surgical site infections are among the most common postoperative complications and remain a major global health concern, further intensified by increasing antimicrobial resistance. Plasma-activated water (PAW) has emerged as a novel antimicrobial strategy, though earlier studies suggested its activity depends on an acidic pH. To overcome this limitation, we previously developed a high-strength, neutral pH-buffered formulation (hs-PAbW) optimized for human clinical use, which preserved strong antimicrobial properties. In the present work, we created a hydrogel incorporating hs-PAbW at a bactericidal yet non-cytotoxic concentration (hs-PAbW-20) and evaluated its therapeutic performance in mouse wound models. In Balb/c mice, hs-PAbW-20 gel markedly reduced Pseudomonas aeruginosa and Staphylococcus aureus loads, demonstrating efficacy comparable to neomycin and soframycin. The gel also enhanced wound closure by regulating key genes involved in tissue repair. In a diabetic C57BL/6 model, hs-PAbW-20 gel similarly lowered P. aeruginosa burden and accelerated healing. Collectively, these results highlight hs-PAbW-20 gel as a promising next-generation wound therapy capable of addressing the shortcomings of traditional antibiotic-based treatments.

microbiology↗

bioRxiv2025

LuxS-mediated AI-2 signaling regulates chemotaxis, flagellar motility, and virulence genes in Salmonella Typhimurium and contributes to colonization in the murine gut

The cooperative response through AI-2 allows Salmonella to coordinate and regulate its community behaviours crucial for its survival and infection. Understanding these mechanisms is essential for developing strategies to interfere with Salmonellas ability to cause disease. In this study, we report that Salmonella Typhimurium uses AI-2 signaling to modulate the expression of chemotaxis and motility-associated genes and the formation of flagella on the bacterial cell surface. The AI-2 acts as a chemoattractant, and the cascade mediates the chemotactic response of Salmonella. Such regulations assist in the attachment and invasion of Salmonella into intestinal epithelial cells both in vitro and in a murine model. Furthermore, we determined that HilD, known to regulate motility by induction of a chemoreceptor and SPI-1 in Salmonella, along with key SPI-1 genes, is underexpressed in the absence of LuxS/AI-2 signaling, which unravels a complex regulatory network by AI-2 in Salmonella. Beyond the initial phase, this signaling facilitates survival in the presence of polymyxin B and supports intracellular survival and persistence within epithelial cells by regulating the pmrD/AB system, thereby evading the antimicrobial peptide immune response. Additionally, our findings show that Salmonella activates its LuxS/AI-2 signalling cascade within the Caco-2 epithelial cells. Complimentary observation through transcriptomic profiling revealed that LuxS/AI-2 signalling governs the coordinated regulation of genes implicated in distinct phases of Salmonella pathogenesis. Lastly using a mouse model, we show that AI-2 signaling is critical for organ colonization and virulence of Salmonella, and inhibition of LuxS/AI-2 signaling in combination with antibiotics can be an alternative therapeutic approach. Thus, our study dissects an integrated regulatory mechanism by which AI-2 signaling mediates critical processes during multiple stages of Salmonella Typhimurium pathogenesis.

microbiology↗

bioRxiv2025

Intracellular formate is a critical mediator of the antimicrobial fate of Salmonella Typhimurium Against meropenem and ciprofloxacin

Salmonella enterica serovar Typhimurium (STM) employs various strategies to endure antibiotic stress, but the contribution of central metabolites to resistance remains insufficiently characterized. In this work, we identify intracellular formate, produced by pyruvate-formate lyase (PflB), as a key determinant of STM susceptibility to meropenem and ciprofloxacin. Deletion of pflB disrupts cellular pH homeostasis, impairing efflux pump function, increasing reactive oxygen species, and causing membrane depolarization, collectively heightening sensitivity to antibiotics. Supplementing extracellular formate reverses these phenotypes. Mechanistically, pH imbalance activates the RpoE-csrB regulatory pathway, leading to reduced expression of efflux pump genes acrB and tolC, while extracellular formate engages the BarA/SirA two-component system to further influence the CsrA/csrB network. Disrupting the formate pool interferes with efflux-dependent resistance, revealing a previously unrecognized signalling function for intracellular metabolites. Overall, our results position formate as a metabolic driver of antibiotic outcomes in STM and highlight it as a potential target for mitigating resistance.

microbiology↗

bioRxiv2025

Maltose facilitates Salmonella growth under nitrogen stress while impairing adhesion to epithelial cells

The composition of nutrients in the intestine defines a niche for colonising gut pathogens. The lack of nutrients suitable for pathogens due to competition from resident intestinal microbiota or dietary preferences leads to colonisation failure by pathogens. In this study, we investigated the impact of the disaccharide sugar maltose on Salmonella Typhimurium (STM) pathogenicity during the early phases of infection in C57BL/6 mice and human colon carcinoma cells (Caco-2). We found that supplementation of maltose at lower concentrations inhibited STM colonisation in the ileum of mice. To understand this, we investigated the role of maltose metabolism in human colon epithelial (Caco-2) cells. Deleting the maltose metabolism gene (malQ) increased adhesion to Caco-2 cells. The increased adhesion was due to increased expression of type 1 fimbriae. Inhibiting the type 1 fimbriae-mediated binding to host epithelial cells by incubating with mannose resulted in similar adhesion of STM WT and STM {Delta}malQ. We further identified that malQ regulates the adhesion of Salmonella through the sigma factor RpoE. Overall, malQ in Salmonella inhibits infection in epithelial cells by reducing its adhesion to host epithelial cells.

microbiology↗

bioRxiv2025

Undecaprenyl Pyrophosphate Phosphatase (UppP) is a pivotal element in Salmonella Intramacrophage Survival

To establish infection, Salmonella confronts a dynamic barrage of host-induced stresses. The peptidoglycan layer, essential for maintaining cell integrity and counteracting environmental stress, relies on the lipid carrier undecaprenyl phosphate, generated by Undecaprenyl pyrophosphate phosphatase (UppP). While UppP is linked to virulence in other pathogens, its role in Salmonella remains unclear. We show that an uppP mutant in S. Typhimurium exhibits altered cell morphology, reduced stiffness, and impaired survival in RAW 264.7 macrophages. The mutant is also attenuated in systemic infection in C57BL/6 mice. These defects are associated with increased sensitivity to nitrosative stress. Notably, iNOS inhibition or deficiency restores intracellular survival of the uppP mutant in both RAW 264.7 macrophages and the mouse model, implicating UppP in resistance to nitrosative stress. Our findings reveal a critical role for UppP in promoting Salmonella survival within macrophages and contributing to systemic pathogenesis.

microbiology↗

bioRxiv2025

LuxS/AI-2 signaling in Salmonella Typhimurium regulates phoP/phoQ to protect against acidic pH for enhanced survival in macrophages

The intestinal milieu is largely characterized by the complex array of chemical compounds produced through the metabolic activity of resident microbiota. Enteric pathogens like Salmonella, which have evolved refined mechanisms to persist within this environment, utilize these microbial metabolites and self-produce quorum molecules as molecular cues to identify ecological niches and modulate their survival and virulence strategies. Salmonella quorum sensing involves producing and detecting Autoinducer-2 (AI-2) signaling molecules. Our research reveals that Salmonella Typhimurium enhances AI-2 biosynthesis and transport under acidic conditions, aiding environmental adaptation and facilitating pathogenesis in macrophages. AI-2 signaling regulates the pH-sensing two-component system genes, phoP/phoQ, ensuring cytosolic pH homeostasis, survival, and acid tolerance. It also involves regulating the lysine/cadaverine-mediated acid tolerance response and maintaining bacterial cytosolic pH. Furthermore, we show that the repressor LsrR protein, apart from the lsr promoter, binds to only one strand of the phoP promoter via its Y25 and R43 amino acids to negatively modulate phoP expression. Additionally this signaling ameliorates the intracellular survival by modulating Salmonella Pathogenicity Island-2 (SPI-2) regulators (ssrA/ssrB) and SPI-2 effector expression via PhoP. Mouse models demonstrate that AI-2 signaling is essential for colonizing the primary and secondary infection sites, showcasing its critical role in pathogenesis and low pH survival mechanisms in Salmonella.

microbiology↗

bioRxiv2024

Evaporation and pathogenesis of levitated bacteria-laden surrogate respiratory fluid droplets: At different relative humidity and evaporation stages

HypothesisAerosols are the principal cause of airborne infections and respiratory diseases. Droplets ejected from the host can evaporate and form a precipitate in the air (aerosol mode), or evaporate for some time, and fall on the ground (mixed mode) or directly fall on the ground and evaporate as sessile mode. Different evaporation modes, stages of evaporation and the relative humidity (RH) conditions affect the survival and infectivity of the bacteria in the precipitate. ExperimentsWe have investigated three droplet diameter reduction ratio-based stages of evaporation of a bacteria-laden levitated droplet at two different RH settings and evaporation modes (aerosol and mixed) mimicking real-life scenarios. The low RH condition mimics evaporation in arid regions. e.g., Delhi and the high RH conditions imitate cold areas like London. The study analyses the mass transport, micro-characterizes the samples, and investigates the survival and infectivity of bacteria in the sample. FindingsThe bacteria survive more in the high RH condition than in the low RH condition for all diameter reduction ratio-based stages and modes of evaporation. For the aerosol mode, at a fixed RH condition, the evaporation time plays a vital role as the bacteria in early-stage partially dried samples are more viable than the full precipitate. The evaporation rate, and the generation of reactive oxygen species (ROS) cause a remarkable difference in the viability and infectivity of the bacterial samples. Therefore, our findings report that the evaporation history of an infected droplet is an indispensable factor in determining bacterial viability and subsequent infectivity.

microbiology↗

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

The resilience of Salmonella to bile stress is impaired due to the reduced efflux pump activity mediated by the antioxidant enzyme YqhD

Bile salts play a critical role in modulating the host gut. They have antimicrobial properties wherein they disrupt the bacterial membrane and produce reactive oxygen species, causing DNA damage. Bile-resistant pathogen like Salmonella regulates their metabolic activity to counteract the effects of bile. This study explores the role of YqhD, a putative alcohol dehydrogenase, in Salmonellas bile salt susceptibility. We observed increased survival of yqhD mutant in the in vitro studies in LB media with bile, liver cell line HepG2 and C57BL/6 mice on treatment with 8% sodium cholate in the intestine. Bile salts are produced for the digestion of fat. Replacing the chow diet with a high-fat diet (HFD) in mice increased organ burden in C57BL/6 mice of the yqhD mutant. Mutation of yqhD on bile salt exposure leads to increased reactive oxygen species and modulation of antioxidant genes in the bacteria. The oxidative stress of the yqhD mutant is indispensable for improved survival when exposed to bile salt. The addition of antioxidant glutathione in vitro reduced the enhanced growth of the yqhD mutant. Similarly, in the gp91-/- phox mice, there was a reduced organ burden of yqhD mutant on exposure to HFD compared to chow-fed mice. Furthermore, the yqhD mutant increases AcrAB efflux pump activity regulated by RamA/R regulon. TeaserRedox gene yqhD regulates bile salt susceptibility by modulating Salmonellas ROS and efflux pump activity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/625033v1_figa1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@14e30f0org.highwire.dtl.DTLVardef@6f14a0org.highwire.dtl.DTLVardef@b3291dorg.highwire.dtl.DTLVardef@503988_HPS_FORMAT_FIGEXP M_FIG A. STM {Delta}yqhD has less organ burden in the C57BL/6 mice than STM WT, and there was a similar organ burden in gp91phox-/-. On HFD exposure, the organ burden of STM {Delta}yqhD became similar to STM WT in C57BL/6 mice, and there was reduced organ of STM {Delta}yqhD burden when gp91phox-/- were exposed to HFD. B. On bile salt exposure, YqhD in Salmonella reduces ROS in bacteria and expression of RamA, decreasing the AcrAB efflux pump activity and reducing survival. C_FIG

microbiology↗

bioRxiv2024

Salmonella effector SseL induces PD-L1 up-regulation and T cell inactivation via beta-catenin signalling axis

The upregulation of PD-L1 by various pathogens is a recognized strategy to evade the adaptive immune response. Salmonella infection also upregulates PD-L1 levels causing culling of the activated T-cell; however, the underlying mechanism behind this upregulation is not known. Our findings indicate that the upregulation of PD-L1 is through Salmonella pathogenicity island 2 (SPI-2) encoded effectors since PFA-fixed STM WT and STM{Delta}ssaV (which is unable to secrete effector proteins) did not alter PD-L1 levels. We have further investigated the role of the SPI-2 effector SseL (a deubiquitinase known to affect the NF-B pathway) in PD-L1 upregulation. Our study identifies SPI-2 effector SseL to be crucial for upregulating PD-L1 in vitro as well as in vivo murine models. The increase in PD-L1 levels induced by STM WT facilitates colonization in secondary infection sites in C57BL/6 mice, including the liver and spleen, while the STM{Delta}sseL strain exhibits significant colonization defects. Notably, despite the reduced colonization capacity of STM{Delta}sseL, infected mice exhibit earlier mortality associated with heightened inflammation. We further elucidated the molecular mechanism behind PD-L1 upregulation and observed that bacterial effector SseL helps in the stabilization of {beta}-catenin inside the cell. {beta}-catenin thus translocates into the nucleus and directly regulates the transcriptional levels of PD-L1, which is abrogated upon using {beta}-catenin/TCF inhibitor FH535. Collectively, our study elucidates the mechanism by which Salmonella mediates immune suppression through PD-L1 upregulation. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/620790v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@12ba486org.highwire.dtl.DTLVardef@3e8b40org.highwire.dtl.DTLVardef@28546corg.highwire.dtl.DTLVardef@1bea897_HPS_FORMAT_FIGEXP M_FIG Abstract figure: Schematic representation of SseL mediated PDL1 upregulation and further affecting the T cell proliferation C_FIG

immunology↗

bioRxiv2024

Reactive nitrogen species (RNS) and its reaction intermediates with reactive oxygen species (ROS) in pH-neutral high-strength plasma-activated water determines the antimicrobial activity against ESKAPE Pathogens

Plasma-activated water (PAW) is an emerging antimicrobial strategy, though its efficacy has traditionally depended on acidic pH to generate reactive species. To improve clinical and practical applicability, we developed a neutral pH formulation using buffered water--hs-PAbW--and evaluated its antimicrobial activity and mechanism of action against ESKAPE pathogens. hs-PAbW significantly reduced bacterial viability during the exponential growth phase. While reactive oxygen species (ROS) alone were insufficient for bacterial killing, reactive nitrogen species (RNS), particularly peroxynitrite, mediated strong microbicidal effects via nitrotyrosine formation. These effects occurred independently of acidic conditions, demonstrating that antimicrobial activity is retained at neutral pH. Alongside, hs-PAbW effectively reduced bacterial burden on metal surfaces and exhibited superior killing compared to high doses of standard antibiotics, including ceftazidime, ciprofloxacin, and meropenem in vitro. A non-cytotoxic dose of hs-PAbW also reduced bacterial load in infected wounds and accelerated wound closure in vivo. These findings highlight the dual functionality of hs-PAbW in surface sanitation and wound care, and support its potential as a sustainable, broad-spectrum antimicrobial agent for clinical use.

bioengineering↗

bioRxiv2024

Odoribacter splanchnicus mitigates Salmonella-induced gut inflammation and its associated pathogenesis via its secreted bacteriocin

Foodborne pathogens pose a major global health hazard, worsened by drug-resistant strains. Therefore, designing novel therapeutic strategies is crucial. Here, we identified the protective role of gut-commensal Odoribacter splanchnicus (OS) against Salmonella in mice by promoting colonization resistance, preserving gut barrier integrity, and preventing acute inflammation and biofilm formation. In vitro, both OS and their culture supernatant inhibited Salmonella biofilm formation, intracellular proliferation in human intestinal cells, and virulence gene expression. Further, our results depicted that OSs protective role acts over a broad spectrum as it confers protection against Gram-positive, Listeria monocytogenes and Gram-negative, Salmonella Typhimurium. Notably, OS conferred protection even when administered post-infection in mice, highlighting its therapeutic potential. Using several biochemical and proteomics approaches, we characterized key OS-secreted molecules that limit intracellular Salmonella and Listeria replication in human intestinal epithelial cells by regulating key virulence effectors and flagella. Collectively, our study establishes OS as the broad-spectrum protective agent against Salmonella and Listeria infections, with promising therapeutic potential. Author SummaryFoodborne pathogens such as Salmonella and Listeria continue to threaten global health, especially with the rise of antibiotic resistance. In this study, we identify the gut commensal bacterium Odoribacter splanchnicus (OS) as a potent protectant against these pathogens. Using a mouse model, we show that OS enhances colonization resistance, maintains gut barrier integrity, and prevents both inflammation and biofilm formation during infection. Remarkably, OS also limits pathogen proliferation and virulence gene expression in human intestinal cells. These protective effects are mediated by both live OS and its secreted molecules within culture supernatant. Importantly, OS administration post-infection also conferred protection, indicating strong therapeutic potential. Biochemical and proteomic analyses revealed OS-secreted factors that suppress key virulence and motility pathways in pathogens. Our findings establish Odoribacter splanchnicus as a promising broad-spectrum biotherapeutic agent against major foodborne infections. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/609322v4_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@7ea42corg.highwire.dtl.DTLVardef@4c8074org.highwire.dtl.DTLVardef@855350org.highwire.dtl.DTLVardef@120decc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Unravelling the Dynamics of Bacterial-Laden Respiratory Droplets: Epidemiological Transfer from Fomites to Susceptible Host

This study provides the first comprehensive investigation into how pathogen-laden respiratory droplets transfer diseases via inanimate surfaces. Respiratory fluid ejections containing pathogens pose a significant health threat, especially in high-traffic areas like hospitals, public transport, restaurants, and schools. When these droplets dry on surfaces, they form deposits that can transfer pathogens to healthy individuals through contact and can be ingested via the oral or nasal route. The study examined the effects of varying salt and mucin concentrations in respiratory fluid droplets containing Pseudomonas aeruginosa (PA). Results showed that PA viability increased tenfold at elevated mucin concentrations, while changes in salt concentration had minimal impact. Adhesive properties of the deposits were analysed using atomic force spectroscopy and scotch tape test. Pathogen transfer from the deposit to a fingerprint patterned model thumb at different relative humidity (RH) levels was assessed using confocal microscopy, showing significant pathogen transfer at elevated RH. Out of 106 CFU/ml pathogens in deposits, 17% to 38% are potentially transferable, with most of the transfer occurring from the droplets edge deposits. The study also explored evaporation, internal flow, and precipitation dynamics, with deposit and pathogen distribution characterized by optical profilometry, scanning electron microscopy, and confocal microscopy.

microbiology↗

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↗

bioRxiv2024

Weather-related changes in the dehydration of respiratory droplets on surfaces bolster bacterial endurance

HypothesisThe study shows for the first time a fivefold difference in the survivability of the bacterium Pseudomonas Aeruginosa (PA) in a realistic respiratory fluid droplet on fomites undergoing drying at different environmental conditions. For instance, in 2023, the annual average relative humidity (RH) in London (UK) is 71%, whereas in Delhi (India), it is 45%, showing that disease spread from fomites could have a demographic dependence. Respiratory fluid droplet ejections containing pathogens on inanimate surfaces are crucial in disease spread, especially in nosocomial settings. However, the interplay between evaporation dynamics, internal fluid flow and precipitation and their collective influence on the distribution and survivability of pathogens at different environmental conditions are less known. ExperimentsShadowgraphy imaging is employed to study evaporation, and optical microscopy imaging is used for precipitation dynamics. Micro-particle image velocimetry (MicroPIV) measurements reveal the internal flow dynamics. Confocal imaging of fluorescently labelled PA elucidates the bacterial distribution within the deposits. FindingsThe study finds that the evaporation rate is drastically impeded during drying at elevated solutal concentrations, particularly at high RH conditions. MicroPIV shows reduced internal flow under high RH conditions. Evaporation rate influences crystal growth, with delayed efflorescence and extending crystallisation times. PA forms denser peripheral arrangements under high evaporation rates and shows a fivefold increase in survivability under low evaporation rates. These findings highlight the critical impact of environmental conditions on pathogen persistence and disease spread from inanimate surfaces. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/579296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@6b9876org.highwire.dtl.DTLVardef@1e6a18forg.highwire.dtl.DTLVardef@78d308org.highwire.dtl.DTLVardef@66ebe8_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Critical concentration of formate governs the expression of the flagellar and the pathogenicity island-1 genes in Salmonella Typhimurium

Host-derived short-chain fatty acids (SCFAs) are extensively being studied for their role in the virulence and pathogenesis of Salmonella Typhimurium (STM). Formate, an SCFA present in the ileum, functions as a signalling molecule to enhance STM invasion. However, the role of intracellular formate in Salmonella virulence remains poorly understood. To investigate this, we generated knockouts of pflB (pyruvate-formate lyase) and focA (formate transporter). Disruption of formate production through pflB deletion led to reduced flagellation and increased hilA and prgH expression, attributed to elevated intracellular pH and membrane damage. This suppression of flagellar machinery drives a shift from adhesion to invasion, regulated by RpoE via the CsrA/csrB pathway. Additionally, we demonstrate that upon compensation for intracellular deficiency of formate, STM{Delta} pflB starts to utilize formate as a signalling molecule to regulate downstream processes. To the best of our knowledge, this study is the first to establish the critical role of the pflB gene in maintaining intracellular pH and controlling virulence gene expression in STM. Lastly, our findings emphasize the importance of modulating pflB expression across intestinal regions to optimize STM invasion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/577997v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1fc5ec3org.highwire.dtl.DTLVardef@1b15b61org.highwire.dtl.DTLVardef@e657caorg.highwire.dtl.DTLVardef@8db00f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗