Search Results (42)

The present Perspective analyzes the remarkable evolution of the Amyloid Cascade Hypothesis 2.0 (ACH2.0) theory of Alzheimer’s disease (AD) since its inception a few years ago, as reflected in the diminishing role of amyloid-beta (Aβ) in the disease. In the initial iteration of the ACH2.0, Aβ-protein-precursor (AβPP)-derived intraneuronal Aβ (iAβ), accumulated to neuronal integrated stress response (ISR)-eliciting levels, triggers AD. The neuronal ISR, in turn, activates the AβPP-independent production of its C99 fragment that is processed into iAβ, which drives the disease. The second iteration of the ACH2.0 stemmed from the realization that AD is, in fact, a disease of the sustained neuronal ISR. It introduced two categories of AD—conventional and unconventional—differing mainly in the manner of their causation. The former is caused by the neuronal ISR triggered by AβPP-derived iAβ, whereas in the latter, the neuronal ISR is elicited by stressors distinct from AβPP-derived iAβ and arising from brain trauma, viral and bacterial infections, and various types of inflammation. Moreover, conventional AD always contains an unconventional component, and in both forms, the disease is driven by iAβ generated independently of AβPP. In its third, the current, iteration, the ACH2.0 posits that proteolytic production of Aβ is suppressed in AD-affected neurons and that the disease is driven by C99 generated independently of AβPP. Suppression of Aβ production in AD seems an oxymoron: Aβ is equated with AD, and the later is inconceivable without the former in an ingrained Amyloid Cascade Hypothesis (ACH)-based notion. But suppression of Aβ production in AD-affected neurons is where the logic leads, and to follow it we only need to overcome the inertia of the preexisting assumptions. Moreover, not only is the generation of Aβ suppressed, so is the production of all components of the AβPP proteolytic pathway. This assertion is not a quantum leap (unless overcoming the inertia counts as such): the global cellular protein synthesis is severely suppressed under the neuronal ISR conditions, and there is no reason for constituents of the AβPP proteolytic pathway to be exempted, and they, apparently, are not, as indicated by the empirical data. In contrast, tau protein translation persists in AD-affected neurons under ISR conditions because the human tau mRNA contains an internal ribosomal entry site in its 5′UTR. In current mouse models, iAβ derived from AβPP expressed exogenously from human transgenes elicits the neuronal ISR and thus suppresses its own production. Its levels cannot principally reach AD pathology-causing levels regardless of the number of transgenes or the types of FAD mutations that they (or additional transgenes) carry. Since the AβPP-independent C99 production pathway is inoperative in mice, the current transgenic models have no potential for developing the full spectrum of AD pathology. What they display are only effects of the AβPP-derived iAβ-elicited neuronal ISR. The paper describes strategies to construct adequate transgenic AD models. It also details the utilization of human neuronal cells as the only adequate model system currently available for conventional and unconventional AD. The final alteration of the ACH2.0, introduced in the present Perspective, is that AβPP, which supports neuronal functionality and viability, is, after all, potentially produced in AD-affected neurons, albeit not conventionally but in an ISR-driven and -compatible process. Thus, the present narrative begins with the “omnipotent” Aβ capable of both triggering and driving the disease and ends up with this peptide largely dislodged from its pedestal and retaining its central role in triggering the disease in only one, although prevalent (conventional), category of AD (and driving it in none). Among interesting inferences of the present Perspective is the determination that “sporadic AD” is not sporadic at all (“non-familial” would be a much better designation). The term has fatalistic connotations, implying that the disease can strike at random. This is patently not the case: The conventional disease affects a distinct subpopulation, and the basis for unconventional AD is well understood. Another conclusion is that, unless prevented, the occurrence of conventional AD is inevitable given a sufficiently long lifespan. This Perspective also defines therapeutic directions not to be taken as well as auspicious ways forward. The former category includes ACH-based drugs (those interfering with the proteolytic production of Aβ and/or depleting extracellular Aβ). They are legitimate (albeit inefficient) preventive agents for conventional AD. There is, however, a proverbial snowball’s chance in hell of them being effective in symptomatic AD, lecanemab, donanemab, and any other “…mab” or “…stat” notwithstanding. They comprise Aβ-specific antibodies, inhibitors of beta- and gamma-secretase, and modulators of the latter. In the latter category, among ways to go are the following: (1) Depletion of iAβ, which, if sufficiently “deep”, opens up a tantalizing possibility of once-in-a-lifetime preventive transient treatment for conventional AD and aging-associated cognitive decline, AACD. (2) Composite therapy comprising the degradation of C99/iAβ and concurrent inhibition of the neuronal ISR. A single transient treatment could be sufficient to arrest the progression of conventional AD and prevent its recurrence for life. Multiple recurrent treatments would achieve the same outcome in unconventional AD. Alternatively, the sustained reduction/removal of unconventional neuronal ISR-eliciting stressors through the elimination of their source would convert unconventional AD into conventional one, preventable/treatable by a single transient administration of the composite C99/iAβ depletion/ISR suppression therapy. Efficient and suitable ISR inhibitors are available, and it is explicitly clear where to look for C99/iAβ-specific targeted degradation agents—activators of BACE1 and, especially, BACE2. Directly acting C99/iAβ-specific degradation agents such as proteolysis-targeting chimeras (PROTACs) and molecular-glue degraders (MGDs) are also viable options. (3) A circumscribed shift (either upstream or downstream) of the position of transcription start site (TSS) of the human AβPP gene, or, alternatively, a gene editing-mediated excision or replacement of a small, defined segment of its portion encoding 5′-untranslated region of AβPP mRNA; targeting AβPP RNA with anti-antisense oligonucleotides is another possibility. If properly executed, these RNA-based strategies would not interfere with the protein-coding potential of AβPP mRNA, and each would be capable of both preventing and stopping the AβPP-independent generation of C99 and thus of either preventing AD or arresting the progression of the disease in its conventional and unconventional forms. The paper is interspersed with “validation” sections: every conceptually significant notion is either validated by the existing data or an experimental procedure validating it is proposed. Full article

Biofilms are resistant microbial cell aggregates that pose risks to the health and food industries and produce environmental contamination. The accurate and efficient detection and prevention of biofilms are challenging and demand interdisciplinary approaches. This multidisciplinary research reports the application of a deep learning-based artificial intelligence (AI) model for detecting biofilms produced by Pseudomonas aeruginosa with high accuracy. Aptamer DNA-templated silver nanocluster (Ag-NC) was used to prevent biofilm formation, which produced images of the planktonic states of the bacteria. Large-volume bright-field images of bacterial biofilms were used to design the AI model. In particular, we used U-Net with ResNet encoder enhancement to segment biofilm images for AI analysis. Different degrees of biofilm structures can be efficiently detected using ResNet18 and ResNet34 backbones. The potential applications of this technique are also discussed. Full article

Background: Infective endocarditis (IE) is an infectious disease caused by the hematogenous dissemination of bacteria into heart valves. Improving the identification of pathogens that cause IE is important to increase the effectiveness of its therapy and reduce the mortality caused by this pathology. Methods: Ten native heart valves obtained from IE patients undergoing heart valve replacements were analyzed. Bacterial invasion in the heart valves was studied by Gram staining of histological sections. Histopathological changes accompanied with bacterial invasion were studied by immunohistochemical analysis of pan-leukocyte marker CD45, platelet marker CD41, and neutrophil myeloperoxidase. The taxonomic diversity of the bacteria was analyzed using 16S rRNA metabarcoding. Results: Gram staining of the histological sections revealed bacterial cells localized on the atrial surface at the leaflet’s free edge or on the ventricular surface at the leaflet’s base within fibrin deposits in only three of the studied heart valves. Bacterial colonies were co-localized with microthrombi (CD41⁺ cells) containing single leucocytes (CD45⁺ cells), represented by segmented neutrophils. As a result of 16S rRNA metabarcoding, we detected the following bacterial genera: Pseudomonas (70% of the studied heart valves), Roseateles (60%), Acinetobacter (40%), Sphingomonas (40%), Enterococcus (30%), Reyranella (20%), Sphingobium (20%), Streptococcus (20%), Agrobacterium (20%), Ralstonia (10%), and Bacillus (10%). Conclusions: A number of opportunistic microorganisms that could not be detected by routine laboratory tests and were not eliminated during antibiotic therapy were identified in the IE-affected heart valves. The obtained results show the importance of 16S rRNA metabarcoding of heart valves removed due to IE not only as an independent diagnostic method but also as a highly accurate approach that complements routine tests for pathogen identification. Full article

Background/objectives: FtsZ, a eukaryotic tubulin homolog and an essential component of the bacterial divisome, is the target of numerous antimicrobial compounds as well as proteins and peptides, most of which inhibit FtsZ polymerization dynamics. We previously showed that the Kil peptide from bacteriophage λ inhibits Escherichia coli cell division by disrupting FtsZ ring assembly, and this inhibition requires the presence of the essential FtsZ membrane anchor protein ZipA. Methods: To investigate Kil’s molecular mechanism further, we employed deletions, truncations, and molecular modeling to identify the minimal residues necessary for its activity. Results: Modeling suggested that Kil’s core segment folds into a helix-turn-helix (HTH) structure. Deleting either the C-terminal 11 residues or the N-terminal 5 residues of Kil still allowed the inhibition of E. coli cell division, but removing both termini nearly abolished this activity, indicating that a minimal region within the Kil HTH core is essential for its structure and function. Another Kil-like peptide from a closely related enterobacterial phage also disrupted FtsZ ring assembly and required ZipA for this activity. Consistent with its broader activity against FtsZ, λ Kil was able to efficiently inhibit cell division of a uropathogenic E. coli (UPEC) strain. Conclusions: Understanding the structure and function of Kil and similar peptides can potentially reveal additional ways to target FtsZ for antimicrobial therapies and elucidate how FtsZ functions in bacterial cell division. Full article

To obtain an effective bacterial biocontrol strain against the fungal pathogen Ganoderma pseudoferreum, causing rubber tree red root rot disease, healthy rubber tree tissue from Baisha County, Hainan Province, was selected as the isolation source, and bacterial strains with strong antifungal effects against G. pseudoferreum were screened. The strain was identified by molecular biology, in vitro root segment tests, pot growth promotion tests, and genome detection. The strain was further evaluated by biological function tests, genome annotation analysis, and plant defense-related enzyme activity detection. The results show that strain LSR7 had good antagonistic effects against G. pseudoferreum, and the inhibition rate reached 88.49%. The strain LSR7 was identified as Bacillus velezensis by genome sequencing. In a greenhouse environment, LSR7 prevents and treats red root rot disease in rubber trees and promotes the growth of rubber tree seedlings. LSR7 secreted cell wall hydrolases (protease, glucanase, and cellulase), amylases, and siderophores. LSR7 also formed biofilms, facilitating plant colonization. Genome prediction showed that LSR7 secreted multiple antifungal lipopeptides. LSR7 enhanced rubber tree resistance to G. pseudoferreum by increasing the activity of defense enzymes. Bacillus velezensis LSR7 has biocontrol potential and is a candidate strain for controlling red root rot disease in rubber trees. Full article

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds. Full article

From the first isolation of the cystovirus bacteriophage Φ6 from Pseudomonas syringae 50 years ago, we have progressed to a better understanding of the structure and transformations of many parts of the virion. The three-layered virion, encapsulating the tripartite double-stranded RNA (dsRNA) genome, breaches the cell envelope upon infection, generates its own transcripts, and coopts the bacterial machinery to produce its proteins. The generation of a new virion starts with a procapsid with a contracted shape, followed by the packaging of single-stranded RNA segments with concurrent expansion of the capsid, and finally replication to reconstitute the dsRNA genome. The outer two layers are then added, and the fully formed virion released by cell lysis. Most of the procapsid structure, composed of the proteins P1, P2, P4, and P7 is now known, as well as its transformations to the mature, packaged nucleocapsid. The outer two layers are less well-studied. One additional study investigated the binding of the host protein YajQ to the infecting nucleocapsid, where it enhances the transcription of the large RNA segment that codes for the capsid proteins. Finally, I relate the structural aspects of bacteriophage Φ6 to those of other dsRNA viruses, noting the similarities and differences. Full article

Obesity and Western-like diet consumption leads to gut microbiome dysbiosis, which is associated with the development of cardio-metabolic diseases and poor health outcomes. The objective of this study was to reduce Western diet-mediated gut microbial dysbiosis, metabolic dysfunction, and systemic inflammation through the administration of a novel combined intervention strategy (oral probiotic bacteria supplements and muscadine grape extract (MGE)). To do so, adult female C57BL/6 mice were fed a low-fat control or Western-style diet and sub-grouped into diet alone, probiotic intervention, antibiotic treatments, MGE supplementation, a combination of MGE and probiotics, or MGE and antibiotics for 13 weeks. Mouse body weight, visceral adipose tissue (VAT), liver, and mammary glands (MG) were weighed at the end of the study. Fecal 16S rRNA sequencing was performed to determine gut bacterial microbiome populations. Collagen, macrophage, and monocyte chemoattractant protein-1 (MCP-1) in the VAT and MG tissue were examined by immunohistochemistry. Adipocyte diameter was measured in VAT. Immunohistochemistry of intestinal segments was used to examine villi length, muscularis thickness, and goblet cell numbers. We show that dietary interventions in Western diet-fed mice modulated % body weight gain, visceral adiposity, MG weight, gut microbial populations, and inflammation. Intervention strategies in both diets effectively reduced VAT and MG fibrosis, VAT and MG macrophages, adipocyte diameter, and VAT and MG MCP-1. Interventions also improved intestinal health parameters. In conclusion, dietary intervention with MGE and probiotics modulates several microbial, inflammatory, and metabolic factors reducing poor health outcomes associated with Western diet intake. Full article

Pathogenic enterotoxigenic Escherichia coli (ETEC) is a major cause of bacterial diarrhea in weaning piglets, which are vulnerable to changes in environment and feed. This study aimed to determine the effects of the ETEC challenge on piglet growth performance, diarrhea rate, jejunal microbial profile, jejunal morphology and goblet cell distribution. A total of 13 piglets from one litter were selected on postnatal day 21 and assigned to treatments with or without ETEC challenge at 1 × 10⁸ CFUs, as ETEC group or control group, respectively. On postnatal day 28, samples were collected, followed by the detection of serum biochemical indexes and inflammatory indicators, HE staining, PAS staining and 16S rDNA gene amplicon sequencing. Results showed that the growth performance decreased, while the diarrhea rate increased for the ETEC group. The jejunum is the main segment of the injured intestine during the ETEC challenge. Compared with the control, the ETEC group displayed fewer goblet cells in the jejunum, where goblet cells are more distributed at the crypt and less distributed at the villus. In addition, ETEC piglets possessed higher abundances of the genus Desulfovibrio, genus Oxalobacter and genus Peptococus and lower abundances of the genus Prevotella 2, genus Flavonifractor and genus Blautra. In terms of alpha diversity, Chao 1 and observed features indexes were both increased for the ETEC group. Our study provides insights into jejunal histopathological impairment and microbial variation in response to ETEC infection for weaned piglets and is a valuable reference for researchers engaged in animal health research to select stress models. Full article

Pediatric osteoarticular infections (OAIs) are serious conditions that can lead to severe septic complications, prolonged morbidity with long-term impaired function, and perturbed subsequent bone development. Kingella kingae (K. kingae) is currently accepted as the predominant pathogen in pediatric OAIs, especially among 6–48 month olds. The present study aimed to identify clinical and biological markers that would refine the detection of patients with an OAI due to K. kingae. We retrospectively studied every consecutive case of pediatric OAI admitted to our institution over 17 years. Medical records were examined for patient characteristics such as temperature at admission, affected segment, and biological parameters such as white blood cell (WBC) count, left shift, platelet count (PLT), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). The 247 patients included 52.2% males and 47.8% females and mean age was 18.5 ± 10 months old. Four patients were older than 48 months; none were younger than 6 months old. Mean temperature at admission was 37.4 ± 0.9 °C. Regarding biological parameters, mean WBC count was 12,700 ± 4180/mm³, left shift was only present in one patient, mean PLT was 419,000 ± 123,000/mm³, mean CRP was 26.6 ± 27.8 mg/L, and mean ESR was 35.0 ± 18.9 mm/h. Compared to the modified predictors of OAI defined by Kocher and Caird, 17.2% of our cases were above their cut-off values for temperature, 52.3% were above the WBC cut-off, 33.5% were above the ESR cut-off, and 46.4% were above the CRP cut-off. OAIs due to K. kingae frequently remain undetected using the classic biological parameters for investigating bacterial infections. As an addition to the predictors normally used (°C, WBC, CRP, and ESR), this study found that elevated platelet count was frequently present during OAIs caused by K. kingae. Although this biological characteristic was inconstant, its presence was highly significant and very suggestive of an invasive infection due to K. kingae. Full article

In this study, we applied bacterial artificial chromosome (BAC) technology with PRV^ΔTK/gE/gI as the base material to replace the first, central, and terminal segments of the US3 gene with codon-deoptimized fragments via two-step Red-mediated recombination in E. coli GS1783 cells. The three constructed BACs were co-transfected with gI and part of gE fragments carrying homologous sequences (gI+gE’), respectively, in swine testicular cells. These three recombinant viruses with US3 codon de-optimization ((PRV^{ΔTK&gE-US3deop−1}, PRV^{ΔTK&gE-US3deop−2}, and PRV^{ΔTK&gE-US3deop−3}) were obtained and purified. These three recombinant viruses exhibited similar growth kinetics to the parental AH02LA strain, stably retained the deletion of TK and gE gene fragments, and stably inherited the recoded US3. Mice were inoculated intraperitoneally with the three recombinant viruses or control virus PRV^ΔTK&gEAH02 at a 10^7.0 TCID₅₀ dose. Mice immunized with PRV^{ΔTK&gE-US3deop−1} did not develop clinical signs and had a decreased virus load and attenuated pathological changes in the lungs and brain compared to the control group. Moreover, immunized mice were challenged with 100 LD₅₀ of the AH02LA strain, and PRV^{ΔTK&gE-US3deop−1} provided similar protection to that of the control virus PRV^ΔTK&gEAH02. Finally, PRV^{ΔTK&gE-US3deop−1} was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 10^6.0 TCID₅₀. Immunized piglets showed only slight temperature reactions and mild clinical signs. However, high levels of seroneutralizing antibody were produced at 14 and 21 days post-immunization. In addition, the immunization of PRV^{ΔTK&gE-US3deop−1} at a dose of 10^5.0 TCID₅₀ provided complete clinical protection and prevented virus shedding in piglets challenged by 10^6.5 TCID₅₀ of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRV^{ΔTK&gE-US3deop−1} displays great potential as a vaccine candidate. Full article

Synthetic surgical meshes are commonly used in abdominal wall reconstruction surgeries to strengthen a weak abdominal wall. Common mesh-related complications include local infection and inflammatory processes. Because cannabigerol (CBG) has both antibacterial and anti-inflammatory properties, we proposed that coating VICRYL (polyglactin 910) mesh with a sustained-release varnish (SRV) containing CBG would prevent these complications. We used an in vitro infection model with Staphylococcus aureus and an in vitro inflammation model of lipopolysaccharide (LPS)-stimulated macrophages. Meshes coated with either SRV-placebo or SRV-CBG were exposed daily to S. aureus in tryptic soy medium (TSB) or macrophage Dulbecco’s modified eagle medium (DMEM). Bacterial growth and biofilm formation in the environment and on the meshes were assessed by changes in optical density, bacterial ATP content, metabolic activity, crystal violet staining, spinning disk confocal microscopy (SDCM), and high-resolution scanning electron microscopy (HR-SEM). The anti-inflammatory effect of the culture medium that was exposed daily to the coated meshes was analyzed by measuring the release of the cytokines IL-6 and IL-10 from LPS-stimulated RAW 264.7 macrophages with appropriate ELISA kits. Additionally, a cytotoxicity assay was performed on Vero epithelial cell lines. We observed that compared with SRV-placebo, the segments coated with SRV-CBG inhibited the bacterial growth of S. aureus in the mesh environment for 9 days by 86 ± 4% and prevented biofilm formation and metabolic activity in the surroundings for 9 days, with respective 70 ± 2% and 95 ± 0.2% reductions. The culture medium that was incubated with the SRV-CBG-coated mesh inhibited LPS-induced secretion of IL-6 and IL-10 from the RAW 264.7 macrophages for up to 6 days without affecting macrophage viability. A partial anti-inflammatory effect was also observed with SRV-placebo. The conditioned culture medium was not toxic to Vero epithelial cells, which had an IC₅₀ of 25 µg/mL for CBG. In conclusion, our data indicate a potential role of coating VICRYL mesh with SRV-CBG in preventing infection and inflammation in the initial period after surgery. Full article

Quantification of the concentration of particular cellular metabolites reports on the actual utilization of metabolic pathways in physiological and pathological conditions. Metabolite concentration also constitutes the readout for screening cell factories in metabolic engineering. However, there are no direct approaches that allow for real-time assessment of the levels of intracellular metabolites in single cells. In recent years, the modular architecture of natural bacterial RNA riboswitches has inspired the design of genetically encoded synthetic RNA devices that convert the intracellular concentration of a metabolite into a quantitative fluorescent signal. These so-called RNA-based sensors are composed of a metabolite-binding RNA aptamer as the sensor domain, connected through an actuator segment to a signal-generating reporter domain. However, at present, the variety of available RNA-based sensors for intracellular metabolites is still very limited. Here, we go through natural mechanisms for metabolite sensing and regulation in cells across all kingdoms, focusing on those mediated by riboswitches. We review the design principles underlying currently developed RNA-based sensors and discuss the challenges that hindered the development of novel sensors and recent strategies to address them. We finish by introducing the current and potential applicability of synthetic RNA-based sensors for intracellular metabolites. Full article

Intestinal stricture remains one of the most intractable complications in Crohn’s disease (CD), and the involved mechanisms are poorly understood. Accumulating evidence suggests that the gut microbiota contributes to the pathogenesis of intestinal fibrosis. In this study, we investigated specific mucosa-associated microbiota related to intestinal strictures and their role in predicting postoperative disease course. Twenty CD patients who had undergone operative treatments were enrolled and followed up. Intestinal mucosa and full-thickness sections from stenotic and non-stenotic sites were sterilely collected. DNA extraction and bacterial 16s rRNA gene sequencing were conducted. Radiological and histological evaluations were performed to assess fibrosis. Microbial alpha diversity was significantly decreased in stenotic sites (p = 0.009). At the genus level, Lactobacillus, Oscillospira, Subdoligranulum, Hydrogenophaga, Clostridium and Allobaculum were decreased in stenotic segments (p < 0.1). The difference in Oscillospira sp. (stenotic vs. non-stenotic) was negatively correlated with the erythrocyte sedimentation rate (correlation coefficient (CC) −0.432, p = 0.057) and white blood cell count (CC −0.392, p = 0.087) and positively correlated with serum free fatty acids (CC 0.575, p < 0.05). This difference was negatively associated with intestinal fibrosis evaluated by imagological and histological methods (CC −0.511 and −0.653, p < 0.05). Furthermore, CD patients with a higher abundance of Oscillospira sp. in the residual intestine might experience longer remission periods (p < 0.05). The mucosa-associated microbiota varied between stenotic and non-stenotic sites in CD. Most notably, Oscillospira sp. was negatively correlated with intestinal fibrosis and postoperative disease course. It could be a promising biomarker to predict post-operative disease recurrence and a microbial-based therapeutic target. Full article

Quantitative phase imaging (QPI) is a non-invasive, label-free technique used to detect aberrant cell morphologies caused by disease, thus providing a useful diagnostic approach. Here, we evaluated the potential of QPI to differentiate specific morphological changes in human primary T-cells exposed to various bacterial species and strains. Cells were challenged with sterile bacterial determinants, i.e., membrane vesicles or culture supernatants, derived from different Gram-positive and Gram-negative bacteria. Timelapse QPI by digital holographic microscopy (DHM) was applied to capture changes in T-cell morphology over time. After numerical reconstruction and image segmentation, we calculated single cell area, circularity and mean phase contrast. Upon bacterial challenge, T-cells underwent rapid morphological changes such as cell shrinkage, alterations of mean phase contrast and loss of cell integrity. Time course and intensity of this response varied between both different species and strains. The strongest effect was observed for treatment with S. aureus-derived culture supernatants that led to complete lysis of the cells. Furthermore, cell shrinkage and loss of circular shape was stronger in Gram-negative than in Gram-positive bacteria. Additionally, T-cell response to bacterial virulence factors was concentration-dependent, as decreases in cellular area and circularity were enhanced with increasing concentrations of bacterial determinants. Our findings clearly indicate that T-cell response to bacterial stress depends on the causative pathogen, and specific morphological alterations can be detected using DHM. Full article