Cellular Microbiology最新文献

Bacteria that migrate (extravasate) out of the bloodstream during vascular dissemination can cause secondary infections in many tissues and organs, including the brain, heart, liver, joints, and bone with clinically serious and sometimes fatal outcomes. The mechanisms by which bacteria extravasate through endothelial barriers in the face of blood flow-induced shear stress are poorly understood, in part because individual bacteria are rarely observed traversing endothelia in vivo, and in vitro model systems inadequately mimic the vascular environment. To enable the study of bacterial extravasation mechanisms, we developed a transmembrane microfluidics device mimicking human blood vessels. Fast, quantitative, three-dimensional live cell imaging in this system permitted single-cell resolution measurement of the Lyme disease bacterium Borrelia burgdorferi transmigrating through monolayers of primary human endothelial cells under physiological shear stress. This cost-effective, flexible method was 10,000 times more sensitive than conventional plate reader-based methods for measuring transendothelial migration. Validation studies confirmed that B. burgdorferi transmigrate actively and strikingly do so at similar rates under static and physiological flow conditions. This method has significant potential for future studies of B. burgdorferi extravasation mechanisms, as well as the transendothelial migration mechanisms of other disseminating bloodborne pathogens.

Malaria parasites undergo a single phase of sexual reproduction in their complex lifecycle. It involves specialised, sexually committed cells called gametocytes, which develop rapidly into mature gametes and mate upon entering the mosquito midgut. Gamete development is unique, involving unprecedentedly fast replication to produce male gametes. Within ~15 minutes a male gametocyte replicates its ~23 Mb genome three times to produce 8 genomes, segregates these into newly-assembled flagellated gametes and releases them to seek female gametes. Here, for the first time, we use fluorescent labelling of de novo DNA synthesis to follow this process at the whole-cell and single-molecule levels. We make several novel observations, including characterising the origin recognition complex protein Orc1 for the first time in gametocytes, finding that cytokinesis is uncoupled from DNA replication (implying a lack of cell cycle checkpoints), and that the single-molecule dynamics of DNA replication are entirely different from the dynamics in asexual schizogony.

Rheumatoid arthritis (RA) is a systemic inflammatory disorder that can cause destructive joint disease, significant disability, and increased mortality. RA is the most frequent of all chronic inflammatory joint diseases, and its prevalence frequency in Pakistan is 1.6 per thousand people. Different cytokines and receptors were involved in the triggering of RA, including interleukin-6 (ILR-6), major histocompatibility complex (MHC) antigen human leukocyte (HLA-DR) receptor, and CD20. Several studies illustrated RA as an inherent immune response and triggered due to the “shared epitope.” Therefore, the involvement of all these receptors (IL-6, HLA-DR, and CD20) leads to the neurological, ocular, respiratory, cardiac, skin, and hematological manifestations that have been considered a potential therapeutic target for drug design. Various herbal, natural, and synthetic source inhibitors of interleukin-6 (IL-6), human leukocyte (HLA-DR), and CD20 were studied and reported previously. Reported inhibitors are compared to elucidate the best inhibitor for clinical trials, leading to the orally active drug. In this study, a computer-aided drug designing approach disclosed the potential inhibitors for all receptors based on their distinct binding affinity. Moreover, drug suitability was carried out using Lipinski’s rule by considering the adsorption, distribution, metabolism, and excretion (ADME) of ligands. Results elucidated “calycosin 7-O-glucoside” and “angeliferulate” as putative ligands for IL-6 and HLA-DR, respectively. However, the pharmacokinetic properties (ADMET) revealed angeliferulate as an effete ligand for the biological system compared to calycosin 7-O-glucoside. Based on docking, drug toxicity profiling or pharmacokinetics, and MD simulation stability, this study highlights orally active therapeutic inhibitors to inhibit the activity of pivotal receptors (IL6, HLA-DR, and CD20) of RA in humans. After clinical trials, the resultant inhibitors could be potential therapeutic agents in the drug development against RA.

Pseudomonas aeruginosa (P.a.) is a major human pathogen capable of causing chronic infections in hosts with weakened barrier functions and host defenses, most notably airway infections commonly observed in individuals with the genetic disorder cystic fibrosis (CF). While mainly described as an extracellular pathogen, previous in vitro studies have described the molecular events leading to P.a. internalization in diverse epithelial cell types. However, the long-term fate of intracellular P.a. remains largely unknown. Here, we developed a model allowing for a better understanding of long-term (up to 120 h) intracellular bacterial survival in the airway epithelial cell line BEAS-2B. Using a tobramycin protection assay, we characterized the internalization, long-term intracellular survival, and cytotoxicity of the lab strain PAO1, as well as clinical CF isolates, and conducted analyses at the single-cell level using confocal microscopy and flow cytometry techniques. We observed that infection at low multiplicity of infection allows for intracellular survival up to 120 h post-infection without causing significant host cytotoxicity. Finally, infection with clinical isolates revealed significant strain-to-strain heterogeneity in intracellular survival, including a high persistence phenotype associated with bacterial replication within host cells. Future studies using this model will further elucidate the host and bacterial mechanisms that promote P. aeruginosa intracellular persistence in airway epithelial cells, a potentially unrecognized bacterial reservoir during chronic infections.

Innate immunity recognizes microorganisms through certain invariant receptors named pattern recognition receptors (PRRs) by sensing conserved pathogen-associated molecular patterns (PAMPs). Their recognition activates several signaling pathways that lead the transcription of inflammatory mediators, contributing to trigger a very rapid inflammatory cascade aiming to contain the local infection as well as activating and instructing the adaptive immunity in a specific and synchronized immune response according to the microorganism. Inflammation is a coordinated process involving the secretion of cytokines and chemokines by macrophages and neutrophils leading to the migration of other leukocytes along the endothelium into the injured tissue. Sustained inflammatory responses can cause deleterious effects by promoting the development of autoimmune disorders, allergies, cancer, and other immune pathologies, while weak signals could exacerbate the severity of the disease. Therefore, PRR-mediated signal transduction must be tightly regulated to maintain host immune homeostasis. Innate immunity deficiencies and strategies deployed by microbes to avoid inflammatory responses lead to an altered immune response that allows the pathogen to proliferate causing death or uncontrolled inflammation. This review analyzes the complexity of the immune response at the beginning of the disease focusing on COVID-19 disease and the importance of unraveling its mechanisms to be considered when treating diseases and designing vaccines.

Malaria is a mosquito-borne infectious disease, caused by unicellular Apicomplexan protozoa of the genus Plasmodium. The sexual stage of Plasmodium is one of the most fascinating aspects of the Plasmodium life cycle, yet relatively less explored until now. The production of sexually fit gametocytes through gametocytogenesis is essential to the transmission of the Plasmodium parasite into an anopheline mosquito vector. Understanding how gametocytogenesis is regulated promotes the identification of novel drug targets and also the development of transmission-blocking vaccines that would help reduce the disease burden in endemic areas. Transcriptional regulation in Plasmodium parasites is primarily controlled by a family of twenty-seven Apicomplexan Apetela 2 (ApiAP2) genes which act in a cascade to enable the parasite to progress through its asexual replication as well as gametocytogenesis. Here, we review the latest progress made on members of the ApiAP2 family characterized as key players of the transcriptional machinery of gametocytes. Further, we will highlight the transcriptional regulation network of ApiAP2 genes at each stage of gametocytogenesis.

The application of molecular techniques to accurately identify protozoan species can correct previous misidentifications based on traditional morphological identification. Colpodea ciliates have many toxicological and cytological applications, but their subtle morphological differences and small body size hinder species delineation. Herein, we used Cox I and β-tubulin genes, alongside fluorescence in situ hybridization (FISH), to evaluate each method in delineating Colpodea species. For this analysis, Colpoda harbinensis n. sp., C. reniformis, two populations of C. inflata, Colpoda compare grandis, and five populations of Paracolpoda steinii, from the soil in northeastern China, were used. We determined that (1) the Cox I gene was more suitable than the β-tubulin gene as a molecular marker for defining intra- and interspecific level relationships of Colpoda. (2) FISH probes designed for Colpoda sp., C. inflata, Colpoda compare grandis, and Paracolpoda steinii, provided rapid interspecific differentiation of Colpodea species. (3) Colpoda harbinensis n. sp. was established and mainly characterized by its size in vivo (approximately 80 × 60 μm), a reniform body in outline, one macronucleus, its spherical shape, a sometimes nonexistent micronucleus, 11–15 somatic kineties, and five or six postoral kineties. In conclusion, combining oligonucleotide probes, DNA barcoding, and morphology for the first time, we have greatly improved the delineation of Colpodea and confirmed that Cox I gene was a promising DNA barcoding marker for species of Colpodea, and FISH could provide useful morphological information as complementing traditional techniques such as silver carbonate.

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen causing invasive disease, including community-acquired bacteraemia, and remains a leading cause of global mortality. Understanding the role of phagocytes in killing bacteria is still limited, especially in vivo. In this study, we established a zebrafish model to study the interaction between intravenously administered pneumococci and professional phagocytes such as macrophages and neutrophils, to unravel bacterial killing mechanisms employed by these immune cells. Our model confirmed the key role of polysaccharide capsule in promoting pneumococcal virulence through inhibition of phagocytosis. Conversely, we show pneumococci lacking a capsule are rapidly internalised by macrophages. Low doses of encapsulated S. pneumoniae cause near 100% mortality within 48 hours postinfection (hpi), while 50 times higher doses of unencapsulated pneumococci are easily cleared. Time course analysis of in vivo bacterial numbers reveals that while encapsulated pneumococcus proliferates to levels exceeding 10⁵ CFU at the time of host death, unencapsulated bacteria are unable to grow and are cleared within 20 hpi. Using genetically induced macrophage depletion, we confirmed an essential role for macrophages in bacterial clearance. Additionally, we show that upon phagocytosis by macrophages, phagosomes undergo rapid acidification. Genetic and chemical inhibition of vacuolar ATPase (v-ATPase) prevents intracellular bacterial killing and induces host death indicating a key role of phagosomal acidification in immunity to invading pneumococci. We also show that our model can be used to study the efficacy of antimicrobials against pneumococci in vivo. Collectively, our data confirm that larval zebrafish can be used to dissect killing mechanisms during pneumococcal infection in vivo and highlight key roles for phagosomal acidification in macrophages for pathogen clearance.

Purpose. To observe the protective effect of homeobox D10 (HOXD10) on acute kidney injury (AKI) by regulating PI3K/AKT signaling pathway is the purpose of this study.Methods. 30 rats were randomly divided into three groups: blank control group, model group, and HOXD10 interference group. The kidney function indexes, HOXD10 protein expression, histopathological features, tubulointerstitial injury, and PI3K and AKT protein expression levels of the three groups were analyzed. Results. Compared with the blank control group, the kidney weight, BUN and SCr in model group increased significantly, and TIL score was higher (P > 0.05). The expression of HOXD10 in model group and HOXD10 interference group were higher than blank control group, and the expression of HOXD10 in HOXD10 interference group was lower than model group (P < 0.05). After we administered HOXD10 blocker to AKI rats, pathological sections by HE staining showed that the kidney tissue damage was significantly reduced compared with the model group, and the expression levels of BUN and SCr in kidney tissue decreased, and the TIL score decreased. The expression of p-PI3K and p-AKT decreased after kidney injury. Compared with the model group, the phosphorylation levels of PI3K and AKT in HOXD10 interference group were significantly increased (P < 0.05). Conclusion. Downregulation of HOXD10 can play a protective role on AKI by activating PI3K/AKT signaling pathway, which can reduce tubulointerstitial injury and improve kidney function.

Invading bacteria can be degraded by selective autophagy, known as xenophagy. Recent studies have shown that the recruitment of autophagy adaptor proteins such as p62 to bacteria and its regulation by activated TANK-binding kinase 1 (TBK1) are required to overcome bacterial infection. However, the detailed molecular mechanisms behind this are not yet fully understood. Here, we show that the human guanylate-binding protein (GBP) family, especially GBP1, directs xenophagy against invading Group A Streptococcus (GAS) by promoting TBK1 phosphorylation. GBP1 exhibits a GAS-surrounding localization response to bacterially caused membrane damage mediated by the membrane damage sensor galectin-3. We found that GBP1 knockout attenuated TBK1 activation, followed by reduced p62 recruitment and lower bactericidal activity by xenophagy. Furthermore, GBP1-TBK1 interaction was detected by immunoprecipitation. Our findings collectively indicate that GBP1 contributes to GAS-targeted autophagy initiated by membrane damage detection by galectin-3 via TBK1 phosphorylation.