Defying the odds: Determinants of the antimicrobial response of Salmonella Typhi and their interplay.

Salmonella Typhi, the invasive serovar of S. enterica subspecies enterica, causes typhoid fever in healthy human hosts. The emergence of antibiotic-resistant strains has consistently challenged the successful treatment of typhoid fever with conventional antibiotics. Antimicrobial resistance (AMR) in Salmonella is acquired either by mutations in the genomic DNA or by acquiring extrachromosomal DNA via horizontal gene transfer. In addition, Salmonella can form a subpopulation of antibiotic persistent (AP) cells that can survive at high concentrations of antibiotics. These have reduced the effectiveness of the first and second lines of antibiotics used to treat Salmonella infection. The recurrent and chronic carriage of S. Typhi in human hosts further complicates the treatment process, as a remarkable shift in the immune response from pro-inflammatory Th1 to anti-inflammatory Th2 is observed. Recent studies have also highlighted the overlap between AP, persistent infection (PI) and AMR. These incidents have revealed several areas of research. In this review, we have put forward a timeline for the evolution of antibiotic resistance in Salmonella and discussed the different mechanisms of the same availed by the pathogen at the genotypic and phenotypic levels. Further, we have presented a detailed discussion on Salmonella antibiotic persistence (AP), PI, the host and bacterial virulence factors that can influence PI, and how both AP and PI can lead to AMR.

SNAREs: a double-edged sword for intravacuolar bacterial pathogens within host cells.

In the tug-of-war between host and pathogen, both evolve to combat each other's defence arsenals. Intracellular phagosomal bacteria have developed strategies to modify the vacuolar niche to suit their requirements best. Conversely, the host tries to target the pathogen-containing vacuoles towards the degradative pathways. The host cells use a robust system through intracellular trafficking to maintain homeostasis inside the cellular milieu. In parallel, intracellular bacterial pathogens have coevolved with the host to harbour strategies to manipulate cellular pathways, organelles, and cargoes, facilitating the conversion of the phagosome into a modified pathogen-containing vacuole (PCV). Key molecular regulators of intracellular traffic, such as changes in the organelle (phospholipid) composition, recruitment of small GTPases and associated effectors, soluble N-ethylmaleimide-sensitive factor-activating protein receptors (SNAREs), etc., are hijacked to evade lysosomal degradation. Legionella, Salmonella, Coxiella, Chlamydia, Mycobacterium, and Brucella are examples of pathogens which diverge from the endocytic pathway by using effector-mediated mechanisms to overcome the challenges and establish their intracellular niches. These pathogens extensively utilise and modulate the end processes of secretory pathways, particularly SNAREs, in repurposing the PCV into specialised compartments resembling the host organelles within the secretory network; at the same time, they avoid being degraded by the host's cellular mechanisms. Here, we discuss the recent research advances on the host-pathogen interaction/crosstalk that involves host SNAREs, conserved cellular processes, and the ongoing host-pathogen defence mechanisms in the molecular arms race against each other. The current knowledge of SNAREs, and intravacuolar bacterial pathogen interactions, enables us to understand host cellular innate immune pathways, maintenance of homeostasis, and potential therapeutic strategies to combat ever-growing antimicrobial resistance.

Observations on phenomenological changes in Klebsiella Pneumoniae under fluidic stresses.

In the present work, experiments are conducted to understand the consequence of stresses generated by flowing fluid on the bacterial morphology and virulence in microfluidic channels. We consider Klebsiella pneumoniae (KP, a clinical isolate), an ESKAPE pathogen, to be the model bacteria responsible for blood stream infections, bacteremia, including pneumonia, urinary tract infections and more. Four different stress conditions are generated 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, the bacterial samples are challenged with ciprofloxacin to see how they behave under different stress conditions. The observations presented in the present study can be extended to model deadly diseases including bacteremia using organ-on-a-chip technology and to understand bacterial pathogenicity under realistic environments.

From Eberthella typhi to Salmonella Typhi: The Fascinating Journey of the Virulence and Pathogenicity of Salmonella Typhi.

Salmonella Typhi (S. Typhi), the invasive typhoidal serovar of Salmonella enterica that causes typhoid fever in humans, is a severe threat to global health. It is one of the major causes of high morbidity and mortality in developing countries. According to recent WHO estimates, approximately 11-21 million typhoid fever illnesses occur annually worldwide, accounting for 0.12-0.16 million deaths. Salmonella infection can spread to healthy individuals by the consumption of contaminated food and water. Typhoid fever in humans sometimes is accompanied by several other critical extraintestinal complications related to the central nervous system, cardiovascular system, pulmonary system, and hepatobiliary system. Salmonella Pathogenicity Island-1 and Salmonella Pathogenicity Island-2 are the two genomic segments containing genes encoding virulent factors that regulate its invasion and systemic pathogenesis. This Review aims to shed light on a comparative analysis of the virulence and pathogenesis of the typhoidal and nontyphoidal serovars of S. enterica.

Absence of proline-peptide transporter YjiY in Salmonella Typhimurium leads to secretion of factors which inhibits intra-species biofilm formation.

Salmonella is a genus of widely spread Gram negative, facultative anaerobic bacteria, which is known to cause »th of diarrheal morbidity and mortality globally. It causes typhoid fever and gastroenteritis by gaining access to the host gut through contaminated food and water. Salmonella utilizes its biofilm lifestyle to strongly resist antibiotics and persist in the host. Although biofilm removal or dispersal has been studied widely, the inhibition of the initiation of Salmonella Typhimurium (STM WT) biofilm remains elusive. This study demonstrates the anti-biofilm property of the cell-free supernatant obtained from a carbon-starvation induced proline peptide transporter mutant (STM ΔyjiY) strain. The STM ΔyjiY culture supernatant primarily inhibits biofilm initiation by regulating biofilm-associated transcriptional network that is reversed upon complementation (STM ΔyjiY:yjiY). We demonstrate that abundance of FlgM correlates with the absence of flagella in the STM ΔyjiY supernatant treated WT cells. NusG works synergistically with the global transcriptional regulator H-NS. Relatively low abundances of flavoredoxin, glutaredoxin, and thiol peroxidase might lead to accumulation of ROS within the biofilm, and subsequent toxicity in STM ΔyjiY supernatant. This work further suggests that targeting these oxidative stress relieving proteins might be a good choice to reduce Salmonella biofilm.

A CcdB toxin-derived peptide acts as a broad-spectrum antibacterial therapeutic in infected mice.

The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S. Typhimurium, S. aureus and A. baumannii. The molecular mechanism likely involves inhibition of Gyrase or Topoisomerase IV, depending on the strain used. The study validates the CcdB binding site on bacterial DNA Gyrase as a viable and alternative target to the fluoroquinolone binding site.

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole.

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 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). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.

Deceiving the big eaters: Salmonella Typhimurium SopB subverts host cell xenophagy in macrophages via dual mechanisms.

Salmonella, a stealthy facultative intracellular pathogen, utilises an array of host immune evasion strategies. This facilitates successful survival via replicative niche establishment in otherwise hostile environments such as macrophages. Salmonella survives in and utilises macrophages for effective dissemination, ultimately leading to systemic infection. Bacterial xenophagy or macro-autophagy is an important host defense mechanism in macrophages. Here, we report for the first time that the Salmonella pathogenicity island-1 (SPI-1) effector SopB is involved in subverting host autophagy via dual mechanisms. SopB is a phosphoinositide phosphatase capable of altering the phosphoinositide dynamics of the host cell. Here, we demonstrate that SopB mediates escape from autophagy by inhibiting the terminal fusion of Salmonella-containing vacuoles (SCVs) with lysosomes and/or autophagosomes. We also report that SopB downregulates overall lysosomal biogenesis by modulating the Akt-transcription factor EB (TFEB) axis via restricting the latter's nuclear localisation. TFEB is a master regulator of lysosomal biogenesis and autophagy. This reduces the overall lysosome content inside host macrophages, further facilitating the survival of Salmonella in macrophages and systemic dissemination of Salmonella.

Profiling system-wide variations and similarities between Rheumatic Heart Disease and Acute Rheumatic Fever-A pilot analysis.

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.

Salmonella Typhimurium PgtE is an essential arsenal to defend against the host resident antimicrobial peptides.

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and occasionally causes systemic infection. Salmonella's ability to survive and replicate within macrophages is an important characteristic during systemic infection. The outer membrane protease PgtE of S. enterica is a member of the Omptin family of outer membrane aspartate proteases which has well-characterized proteolytic activities in-vitro against a wide range of physiologically relevant substrates. However, no study has been done so far that draws a direct correlation between these in-vitro observations and the biology of the pathogen in-vivo. The main goals of this study were to characterize the pathogenesis-associated functions of pgtE and study its role in the intracellular survival and in-vivo virulence of Salmonella Typhimurium. Our study elucidated a possible role of Salmonella Typhimurium pgtE in combating host antimicrobial peptide- bactericidal/ permeability increasing protein (BPI) to survive in human macrophages. The pgtE-deficient strain of Salmonella showed attenuated proliferation and enhanced colocalization with BPI in U937 and Thp1 cells. In the presence of polymixin B, the attenuated in-vitro survival of STM ΔpgtE suggested a role of PgtE against the antimicrobial peptides. In addition, our study revealed that compared to the wild type Salmonella, the pgtE mutant is replication-deficient in C57BL/6 mice. Further, we showed that PgtE interacts directly with several antimicrobial peptides (AMPs) in the host gut. This gives the pathogen a survival advantage and helps to mount a successful infection in the host.

Physics of self-assembly and morpho-topological changes of Klebsiella pneumoniae in desiccating sessile droplets.

HYPOTHESIS: The bacteria suspended in pure water self-assemble into unique patterns depending on bacteria-bacteria, bacteria-substrate and bacteria-liquid interactions. The physical forces acting on bacteria vary based on their respective spatial location inside the droplet cause an assorted magnitude of physical stress. The shear and dehydration induced stress on pathogens(bacteria) in drying bio-fluid droplets alters the viability and infectivity. EXPERIMENTS: We have investigated the flow and desiccation-driven self-assembly of Klebsiella pneumoniae in the naturally evaporating sessile droplets. Klebsiella pneumoniae exhibits extensive changes in its morphology and forms unique patterns as the droplet dries, revealing hitherto unexplored rich physics governing its survival and infection strategies. Self-assembly of bacteria at the droplet contact line is characterized by order-to-disorder packing transitions with high packing densities and excessive deformations (analysed using scanning electron microscopy and atomic force microscopy). In contrast, thin-film instability-led hole formation at the center of the droplet engenders spatial packing of bacteria analogous to honeycomb weathering. FINDINGS: Self-assembly favors the bacteria at the rim of the droplet, leading to enhanced viability and pathogenesis on the famously known "coffee ring" of the droplet compared to the bacteria present at the center of the droplet residue. Mechanistic insights gained via our study can have far-reaching implications for bacterial infection through droplets, e.g., through open wounds.

Salmonella Typhimurium U32 peptidase, YdcP, promotes bacterial survival by conferring protection against in vitro and in vivo oxidative stress.

YdcP, a U32 peptidase, is characterized as a putative collagenase with a role in several bacterial infections. However, its role in the pathogenesis of Salmonella Typhimurium remains elusive. Here, we investigated the role of U32 peptidase, YdcP, in the intracellular survival of S. Typhimurium (STM). Our study revealed a novel function of YdcP in protecting wild-type Salmonella from in vitro and in vivo oxidative stress. The ydcP knockout strain showed attenuated intracellular proliferation within the murine and human macrophages. Incubation of wild-type Salmonella with H2O2 induced the transcript level expression of ydcP. Moreover, deleting ydcP increased the susceptibility of the bacteria to in vitro oxidative stress. STM ΔydcP showed increased colocalization with the gp91phox subunit of the NADPH phagocytic oxidase in RAW264.7 cells. Further, we observed a reduction in the expression of bacterial anti-oxidant genes in STM ΔydcP growing within the RAW264.7 cells. The delay in the death of BALB/c mice infected with STM ΔydcP proved the association of ydcP with the in vivo pathogenesis of Salmonella. Finally, the attenuated growth of the ydcP mutant in wild-type C57BL/6 mice and the recovery of their growth inhibition in gp91phox-/- C57BL/6 mice endorsed the role of ydcP in protecting Salmonella from in vivo oxidative stress. Together, our study depicts a novel role of Salmonella Typhimurium YdcP, a putative U32 peptidase in rendering protection against oxidative stress.

Erratum: Author affiliation addition: "Hepatitis B virus detected in paper currencies in a densely populated city of India: A plausible source of horizontal transmission?"

[This corrects the article on p. 775 in vol. 12, PMID: 33200016.].

Evolving and assembling to pierce through: Evolutionary and structural aspects of antimicrobial peptides.

The burgeoning menace of antimicrobial resistance across the globe has necessitated investigations into other chemotherapeutic strategies to combat infections. Antimicrobial peptides, or host defense peptides, are a set of promising therapeutic candidates in this regard. Most of them cause membrane permeabilization and are a key component of the innate immune response to pathogenic invasion. It has also been reported that peptide self-assembly is a driving factor governing the microbicidal activity of these peptide candidates. While efforts have been made to develop novel synthetic peptides against various microbes, many clinical trials of such peptides have failed due to toxicity and hemolytic activity to the host. A function-guided rational peptide engineering, based on evolutionary principles, physicochemical properties and activity determinants of AMP activity, is expected to help in targeting specific microbes. Furthermore, it is important to develop a unified understanding of the evolution of AMPs in order to fully appreciate their importance in host defense. This review seeks to explore the evolution of AMPs and the physicochemical determinants of AMP activity. The specific interactions driving AMP self-assembly have also been reviewed, emphasizing implications of this self-assembly on microbicidal and immunomodulatory activity.

GH18 family glycoside hydrolase Chitinase A of Salmonella enhances virulence by facilitating invasion and modulating host immune responses.

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.

Multiscale vapor-mediateddendritic pattern formation and bacterial aggregation in complex respiratory biofluid droplets.

HYPOTHESIS: Deposits of biofluid droplets on surfaces (such as respiratory droplets formed during an expiratory) are composed of water-based salt-protein solution that may also contain an infection (bacterial/viral). The final patterns of the deposit formed and bacterial aggregation on the deposits are dictated by the fluid composition and flow dynamics within the droplet. EXPERIMENTS: This work reports the spatio-temporal, topological regulation of deposits of respiratory fluid droplets and control of bacterial aggregation by tweaking flow inside droplets using non-contact vapor-mediated interactions. Desiccated respiratory droplets form deposits with haphazard multiscale dendritic, cruciform-shaped precipitates when evaporated on a glass substrate. However, we showcase that short and long-range vapor-mediated interaction between the droplets can be used as a tool to control these deposits at nano-micro-millimeter scales. We morphologically control hierarchial dendrite size, orientation and subsequently suppress cruciform-shaped crystals by placing a droplet of ethanol in the vicinity of the biofluid droplet. Active living matter in respiratory fluids like bacteria is preferentially segregated and agglomerated without its viability and pathogenesis attenuation. FINDINGS: The nucleation sites can be controlled via preferential transfer of solutes in the droplets; thus, achieving control over crystal occurrence, growth dynamics, and the final topology of the deposit. For the first time, we have experimentally presented a proof-of-concept to control the aggregation of live active matter like bacteria without any direct contact. The methodology can have ramifications in biomedical applications like disease detection and bacterial segregation.

Lipid larceny: channelizing host lipids for establishing successful pathogenesis by bacteria.

Lipids are complex organic compounds made up of carbon, oxygen, and hydrogen. These play a diverse and intricate role in cellular processes like membrane trafficking, protein sorting, signal transduction, and bacterial infections. Both Gram-positive bacteria (Staphylococcus sp., Listeria monocytogenes, etc.) and Gram-negative bacteria (Chlamydia sp., Salmonella sp., E. coli, etc.) can hijack the various host-lipids and utilize them structurally as well as functionally to mount a successful infection. The pathogens can deploy with various arsenals to exploit host membrane lipids and lipid-associated receptors as an attachment for toxins' landing or facilitate their entry into the host cellular niche. Bacterial species like Mycobacterium sp. can also modulate the host lipid metabolism to fetch its carbon source from the host. The sequential conversion of host membrane lipids into arachidonic acid and prostaglandin E2 due to increased activity of cPLA-2 and COX-2 upon bacterial infection creates immunosuppressive conditions and facilitates the intracellular growth and proliferation of bacteria. However, lipids' more debatable role is that they can also be a blessing in disguise. Certain host-lipids, especially sphingolipids, have been shown to play a crucial antibacterial role and help the host in combating the infections. This review shed light on the detailed role of host lipids in bacterial infections and the current understanding of the lipid in therapeutics. We have also discussed potential prospects and the need of the hour to help us cope in this race against deadly pathogens and their rapidly evolving stealthy virulence strategies.

Hepatitis B virus detected in paper currencies in a densely populated city of India: A plausible source of horizontal transmission?

BACKGROUND: The recent rise in the incidence of hepatitis B virus (HBV) infections in a densely populated city of eastern India ("mixing vessel" of people of varied socio-economic and immune status) prompted this study. Applying saliva on fingers for enumerating bank notes is a common practice in the Indian subcontinent. Paper notes may be a potential source of "horizontal" transmission of this virus, especially if there are cuts/bruises on the oral mucous membrane or skin. AIM: To investigate whether paper currencies could be a plausible mode of horizontal transmission of HBV infection. METHODS: Polymerase chain reactions (PCR) followed by nucleotide sequencing was done for the detection of HBV. Hepatitis B virus surface antigen enzyme-linked immunosorbent assay(HBsAg ELISA) was performed on all HBV deoxyribonucleic acid-positive samples to check the detectability of the virus. Atomic force microscopy (AFM) was carried out for visual confirmation of HBV particles in ultracentrifuged/immunoprecipitated samples from currency paper washings. RESULTS: HBV-specific PCRs on pellets obtained after ultracentrifugation/ immunoprecipitation of the currency paper washings detected potentially intact/viable HBV (genotype D2) in 7.14% of samples (n = 70). AFM gave the visual confirmation of HBV particles in ultracentrifuged/immunoprecipitated samples from currency paper washings. However, HBV isolates from the currency notes could not be detected by HBsAg ELISA. CONCLUSION: It is a common practice in the Indian subcontinent to count paper currencies by applying saliva on fingertips. Paper notes may be a potential source of "horizontal" transmission of this virus, especially if there are cuts/bruises on the oral mucous membrane or skin, but it was practically not possible to demonstrate experimentally such transmission. Detection of potentially intact/viable and "occult" HBV from currency poses potential risk of silent transmission of this virus among the general population.

Enteropathogens: Tuning Their Gene Expression for Hassle-Free Survival.

Enteropathogenic bacteria have been the cause of the majority of foodborne illnesses. Much of the research has been focused on elucidating the mechanisms by which these pathogens evade the host immune system. One of the ways in which they achieve the successful establishment of a niche in the gut microenvironment and survive is by a chain of elegantly regulated gene expression patterns. Studies have shown that this process is very elaborate and is also regulated by several factors. Pathogens like, enteropathogenic Escherichia coli (EPEC), Salmonella Typhimurium, Shigella flexneri, Yersinia sp. have been seen to employ various regulated gene expression strategies. These include toxin-antitoxin systems, quorum sensing systems, expression controlled by nucleoid-associated proteins (NAPs), several regulons and operons specific to these pathogens. In the following review, we have tried to discuss the common gene regulatory systems of enteropathogenic bacteria as well as pathogen-specific regulatory mechanisms.