Biology subjects

Rastogi, S. K.

Publications and source records attributed to Rastogi, S. K..

3 recordsLinked to original sources

bioRxiv2024

Acinetobacter baumannii DacC influences cell shape, biofilm formation, and physiological fitness by manifesting DD-carboxypeptidase and β-lactamase dual-enzyme activities

With the growing threat of drug-resistant Acinetobacter baumannii, there is an urgent need to comprehensively understand the physiology of this nosocomial pathogen. As penicillin-binding proteins are attractive targets for antibacterial therapy, herein we have tried to explore the physiological roles of two putative DD-carboxypeptidases, viz., dacC and dacD in A. baumannii. Surprisingly, the deletion of dacC resulted in a reduced growth rate, loss of rod-shaped morphology, reduction in biofilm-forming ability, and enhanced susceptibility towards {beta}-lactams, whereas, the deletion of dacD had no such effect. Interestingly, ectopic expression of dacC restored the lost phenotypes. The double deletion mutant in which both dacC and dacD were absent showed properties similar to the dacC single knockout. On the other hand, cell-shape reverting efficiency in septuple PBP deleted E. coli and in vitro enzyme kinetics assessments reveal that dacD is a stronger DD-CPase as compared to dacC. The expression of dacC was in the log phase whereas dacD expression takes place in the stationary phase. In summary, we conclude that dacC encodes a dual enzyme, possessing activities of DD-CPase and {beta}-lactamase, which significantly affects the physiology of A. baumannii in various ways whereas dacD encodes a strong DD-CPase and plays a role in cell morphology, though it exerts negligible impact on other physiological aspects like intrinsic antibiotic resistance or biofilm formation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/603720v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@18820c9org.highwire.dtl.DTLVardef@208baeorg.highwire.dtl.DTLVardef@b2673eorg.highwire.dtl.DTLVardef@983aae_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

A magnesium transporter CorA of Mycobacterium smegmatis enhances the tolerance of structurally unrelated antibiotics in the host cells

Ion transporters or channels are involved in maintaining metal homeostasis in bacterial cells by aiding the movement of metal ions across the cell, which might also facilitate the export of antimicrobials. Ubiquitous magnesium transporter, CorA of Mycobacterium smegmatis is well known for its role in maintaining magnesium homeostasis. However, little is known about its involvement in exerting antimicrobial resistance. Here, with the help of molecular genetics, in vivo and in silico studies we tried to envisage the role of CorA of M. smegmatis in antimicrobial resistance of M. smegmatis and E. coli. Expression of corA in M. smegmatis and E. coli increased the tolerance of the host cells towards various structurally unrelated antibiotics and anti-tubercular drugs. In addition, a significantly lower accumulation of norfloxacin and ofloxacin by the host cells expressing corA further indicated its role in enhancing the efflux pump activity. Moreover, the presence of a sub-inhibitory concentration of Mg2+ resulted in increased low-level tolerance towards the tested drugs. Furthermore, CorA enhanced the biofilm-forming ability of cells expressing it. Overall, we speculate that magnesium transporter CorA facilitates multi-drug efflux activity of the host cells where Mg2+ might act as a facilitator in the process. IMPORTANCEMagnesium acts as a co-factor for various biochemical and physiological reactions, such as protein synthesis, cell membrane integrity, nucleic acid synthesis, etc. Metal transporters maintain metal homeostasis by regulating the uptake, efflux, or transportation of metals in certain necessary cellular compartments. In bacteria, magnesium ion (Mg2+) is mainly supplied by the CorA protein which is a ubiquitous family of transport proteins and extensively studied in E. coli and Salmonella sp. However, little is known about the functional relationship of metal transporters of Mycobacterium sp with extrusion of antibiotics, and their involvement in stress tolerance. Here, we report CorA (MSMEG_5056), a magnesium transporter of Mycobacterium smegmatis in influencing the extrusion of multiple structurally unrelated classes of drugs and enhancing the biofilm formation of E. coli and Mycobacterium smegmatis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/602764v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@11a5f5corg.highwire.dtl.DTLVardef@6ac8f9org.highwire.dtl.DTLVardef@b083dcorg.highwire.dtl.DTLVardef@a0fb8a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Hypothetical model of Antibiotic export by CorA. Antibiotics bind to the closed state of the protein (left). During the transition to the open state (right), the to-and-fro motion between multiple open states drives the efflux of the antibiotic while facilitating the import of Mg2+. The colors of the models correspond to the chain ID. The bottom views of both the closed and open states are shown in the rectangular box, with the color indicated by their respective chain IDs. C_FIG

microbiology↗

bioRxiv2020

Bioelectrical Interfaces with Cortical Spheroids in Three-Dimensions

Three-dimensional (3D) neuronal spheroid culture serves as a powerful model system for the investigation of neurological disorders and drug discovery. The success of such a model system requires techniques that enable high-resolution functional readout across the entire spheroid. Conventional microelectrode arrays and implantable neural probes cannot monitor the electrophysiology activity across the entire native 3D geometry of the cellular construct. Here, we demonstrate a 3D self-rolled biosensor array (3D-SR-BA) integrated with a 3D cortical spheroid culture for simultaneous in-vitro electrophysiology recording, functional Ca2+ imaging, and drug effect monitoring. We have also developed a signal processing pipeline to detect neural firings with high spatiotemporal resolution from the electrophysiology recordings based on established spike sorting methods. The 3D-SR-BAs cortical spheroid interface provides a stable, high sensitivity recording of neural action potentials (< 50 V peak-to-peak amplitude). The 3D-SR-BA is demonstrated as a potential drug screening platform through the investigation of the neural response to the excitatory neurotransmitter glutamate. Upon addition of glutamate, the neuronal firing rates increased notably corresponding well with the functional Ca2+ imaging. Our entire system, including the 3D-SR-BA integrated with neural spheroid culture, enables simultaneous electrophysiology recording and functional Ca2+ imaging with high spatiotemporal resolution in conjunction with chemical stimulation. We demonstrate a powerful toolset for future studies of tissue development, disease progression, and drug testing and screening, especially when combined with native spheroid cultures directly extracted from humans.

bioengineering↗