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Salmonella is a facultative intracellular pathogen that has co-evolved with its host and has also developed various strategies to evade the host immune responses. Salmonella recruits an array of virulence factors to escape from host defense mechanisms. Previously chitinase A ( chiA ) was found to be upregulated in intracellular Salmonella . Although studies show that several structurally similar chitinases and chitin-binding proteins (CBP) of many human pathogens have a profound role in various aspects of pathogenesis, like adhesion, virulence, and immune evasion, the role of chitinase in the intravacuolar pathogen Salmonella has not yet been elucidated. Therefore, we made chromosomal deletions of the chitinase encoding gene ( chiA ) to study the role of chitinase of Salmonella enterica in the pathogenesis of the serovars, Typhimurium, and Typhi using in vitro cell culture model and two different in vivo hosts. Our data indicate that ChiA removes the terminal sialic acid moiety from the host cell surface, and facilitates the invasion of the pathogen into the epithelial cells. Interestingly we found that the mutant bacteria also quit the Salmonella -containing vacuole and hyper-proliferate in the cytoplasm of the epithelial cells. Further, we found that ChiA aids in reactive nitrogen species (RNS) and reactive oxygen species (ROS) production in the phagocytes, leading to MHCII downregulation followed by suppression of antigen presentation and antibacterial responses. Notably, in the murine host, the mutant shows compromised virulence, leading to immune activation and pathogen clearance. In continuation of the study in C . elegans , Salmonella Typhi ChiA was found to facilitate bacterial attachment to the intestinal epithelium, intestinal colonization, and persistence by downregulating antimicrobial peptides. This study provides new insights on chitinase as an important and novel virulence determinant that helps in immune evasion and increased pathogenesis of Salmonella .

Rheumatic heart disease (RHD) continues to affect developing countries with low income due to the lack of resources and effective diagnostic techniques. Understanding the genetic basis common to both the diseases and that of progression from its prequel disease state, Acute Rheumatic Fever (ARF), would aid in developing predictive biomarkers and improving patient care. To gain system-wide molecular insights into possible causes for progression, in this pilot study, we collected blood transcriptomes from ARF (5) and RHD (5) patients. Using an integrated transcriptome and network analysis approach, we identified a subnetwork comprising the most significantly differentially expressed genes and most perturbed pathways in RHD compared to ARF. For example, the chemokine signaling pathway was seen to be upregulated, while tryptophan metabolism was found to be downregulated in RHD. The subnetworks of variation between the two conditions provide unbiased molecular-level insights into the host processes that may be linked with the progression of ARF to RHD, which has the potential to inform future diagnostics and therapeutic strategies. We also found a significantly raised neutrophil/lymphocyte ratio in both ARF and RHD cohorts. Activated neutrophils and inhibited Natural Killer cell gene signatures reflected the drivers of the inflammatory process typical to both disease conditions.

With the advancements of semantic web, ontology has become the crucial mechanism for representing concepts in various domains. For research and dispersal of customized healthcare services, a major challenge is to efficiently retrieve and analyze individual patient data from a large volume of heterogeneous data over a long time span. This requirement demands effective ontology-based information retrieval approaches for clinical information systems so that the pertinent information can be mined from large amount of distributed data.This unique and groundbreaking book highlights the key advances in ontology-based information retrieval techniques being applied in the healthcare domain and covers the following areas: Semantic data integration in e-health care systems Keyword-based medical information retrieval Ontology-based query retrieval support for e-health implementation

Immortalised cell lines that mimic their primary cell counterparts are fundamental to research, particularly when large cell numbers are required. Here, we report that immortalisation of bone marrow–derived macrophages (iBMDMs) using the J2 virus resulted in the loss of a protein of interest, MSR1, in WT cells by an unknown mechanism. This led us to perform an in-depth mass spectrometry–based proteomic characterisation of common murine macrophage cell lines (J774A.1, RAW264.7, and BMA3.1A7), in comparison with the iBMDMs, as well as primary BMDMs from both C57BL/6 and BALB/c mice. This analysis revealed striking differences in protein profiles associated with macrophage polarisation, phagocytosis, pathogen recognition, and interferon signalling. Among the cell lines, J774A.1 cells were the most similar to the gold standard primary BMDM model, whereas BMA3.1A7 cells were the least similar because of the reduction in abundance of several key proteins related closely to macrophage function. This comprehensive proteomic dataset offers valuable insights into the use and suitability of macrophage cell lines for cell signalling and inflammation research.

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 ΔssaV) but not with SPI-1 encoded T3SS (STM ΔinvC). 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. Salmonella 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.

Fluidic habitats are very common to bacterial life, however, very little is known about the effect of the flow stresses on the virulence of the bacteria. In the present work, we conduct microfluidic experiments to understand the consequence of stresses generated by flowing fluid on the bacterial morphology and virulence. We consider Klebsiella pneumoniae (KP), an ESKAPE pathogen as the model bacteria that are responsible for blood stream infections like bacteremia apart from pneumonia, urinary tract infections and more. We generate four different stress conditions by changing the flow rate and channel geometry subsequently altering the shear rate and stressing time (τ). We observe significant changes in the structural aspects of the stressed bacteria. With an increase in stressing parameters, the viability of the bacterial sample deteriorated. Most importantly, these stressed samples proliferate much more than unstressed samples inside the RAW264.7 murine macrophages. The results shed light on the complex relationship between flow stresses and bacterial virulence. Furthermore, we challenge the bacterial samples with ciprofloxacin to see how they behave under different stress conditions. The present study can be extended to model deadly diseases like bacteremia using organ-on-a-chip technology and help understand bacterial pathogenicity under realistic environments. biorxiv;2023.09.18.558194v1/UFIG1F1ufig1Figure:A schematic representation of the present work. Figure created with BioRender (www.biorender.com)

Deposits of biofluid droplets on surfaces (such as respiratory droplets formed during an expiratory event fallen on surfaces) are composed of the water based salt protein solution that may also contain an infection (bacterial/viral). The final patterns of the deposit formed are dictated by the composition of the fluid and flow dynamics within the droplet. This work reports the spatio temporal, topological regulation of deposits of respiratory fluid droplets and control of motility of bacteria by tweaking flow inside droplets using non contact vapor mediated interactions. When evaporated on a glass surface, respiratory droplets form haphazard multiscale dendritic, cruciform shaped precipitates using vapor mediation as a tool to control these deposits at the level of nano, micro, millimeter scales. We morphologically control dendrite orientation, size and subsequently suppress cruciform-shaped crystals. The nucleation sites are controlled via preferential transfer of solutes in the droplets; thus, achieving control over crystal occurrence and growth dynamics. The active living matter like bacteria is also preferentially segregated with controlled motility without attenuation of its viability and pathogenesis. For the first time, we have experimentally presented a proof of concept to control the motion of live active matter like bacteria in a near nonintrusive manner. The methodology can have ramifications in biomedical applications like disease detection, controlling bacterial motility, and bacterial segregation. Competing Interest Statement The authors have declared no competing interest.

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.Competing Interest StatementThe authors have declared no competing interest.

Macrophages engulf pathogens into dynamic phagosomes, which many bacteria manipulate for survival. However, isolating pure pathogen-containing phagosomes remains challenging. Here, we developed a novel flow cytometry-based isolation and ultrasensitive proteomics approach to analyse phagosomal and bacterial proteomes from macrophages infected with wild-type (WT) Salmonella enterica serovar Typhimurium (STM) or a ΔphoP mutant at 30 min and 4 hrs post-infection. Our approach provides higher throughput, requires lower cell numbers and quantifies more proteins than previous techniques. Our data reveals key host-pathogen interactions, showing induction of PhoP-dependent virulence factors and novel putative proteins that shape STM’s intracellular niche. Moreover, our data indicates that bacteria-containing phagosomes recruit mitochondrial membrane for production of reactive oxygen species. These findings provide new insights into Salmonella’s manipulation of phagosomal maturation and intracellular niche formation.

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. Competing Interest Statement The authors have declared no competing interest.