Intracellular bacteria have evolved mechanisms to invade host cells, establish an intracellular niche that allows survival and replication, produce progeny, and exit the host cell after completion of ...the replication cycle to infect new target cells. Bacteria exit their host cell by (i) initiation of apoptosis, (ii) lytic cell death, and (iii) exocytosis. While bacterial egress is essential for bacterial spreading and, thus, pathogenesis, we currently lack information about egress mechanisms for the obligate intracellular pathogen C. burnetii, the causative agent of the zoonosis Q fever. Here, we demonstrate that C. burnetii inhibits host cell apoptosis early during infection, but induces and/or increases apoptosis at later stages of infection. Only at later stages of infection did we observe C. burnetii egress, which depends on previously established large bacteria‐filled vacuoles and a functional intrinsic apoptotic cascade. The released bacteria are not enclosed by a host cell membrane and can infect and replicate in new target cells. In summary, our data argue that C. burnetii egress in a non‐synchronous way at late stages of infection. Apoptosis‐induction is important for C. burnetii egress, but other pathways most likely contribute.
It is unknown how C. burnetii egresses from its host cell. Here, we demonstrate that C. burnetii inhibits host cell apoptosis early during infection, but induces apoptosis at later stages. Large bacteria‐filled vacuoles and a functional intrinsic apoptotic cascade are a prerequisite for egress in a non‐synchronous way at late stages of infection. Released bacteria are not enclosed by a host cell membrane and can infect and replicate in new target cells.
Salmonella enterica serovar Typhimurium is an invasive, facultative intracellular gastrointestinal pathogen that destroys the brush border of polarized epithelial cells (PEC). The brush border is ...critical for the functions of PEC because it resorbs nutrients from the intestinal lumen and builds a physical barrier to infecting pathogens. The manipuation of PEC during infection by Salmonella was investigated by live-cell imaging and ultrastructural analysed of the brush border. We demonstrate that the destruction of the brush border by Salmonella significantly reduces the resorption surface of PEC along with the abrogation of endocytosis at the apical side of PEC. Both these changes in the physiology of PEC were associated with the translocation of type III secretion system effector protein SopE. Additionally, the F-actin polymerization rate at the apical side of PEC was highly altered by SopE, indicating that reduced endocytosis observed in infected PEC is related to the manipulation of F-actin polymerization mediated by SopE and, to a lesser extent, by effectors SopE2 or SipA. We further observed that in the absence of SopE, Salmonella effaced microvilli and induced reticular F-actin by bacterial accumulation during prolonged infection periods. In contrast to strains translocating SopE, strains lacking SopE did not alter resorption by PEC. Finally, we observed that after engulfment of Salmonella , ezrin was lost from the apical side of PEC and found later in early endosomes containing Salmonella . Our observations suggest that the destruction of the brush border by Salmonella may contribute to the pathogenesis of diarrhea.
Enterocyte invasion by the gastrointestinal pathogen
is accompanied by loss of brush border and massive remodeling of the actin cytoskeleton, leading to microvilli effacement and formation of ...membrane ruffles. These manipulations are mediated by effector proteins translocated by the
Pathogenicity Island 1-encoded type III secretion system (SPI1-T3SS). To unravel the mechanisms of microvilli effacement and contribution of SPI1-T3SS effector proteins, the dynamics of host-pathogen interactions was analyzed using live cell imaging (LCI) of polarized epithelial cells (PEC) expressing LifeAct-GFP. PEC were infected with
wild-type and mutant strains with defined defects in SPI1-T3SS effector proteins, and pharmacological inhibition of actin assembly were applied. We identified that microvilli effacement involves two distinct mechanisms: i) F-actin depolymerization mediated by villin and ii), the consumption of cytoplasmic G-actin by formation of membrane ruffles. By analyzing the contribution of individual SPI1-T3SS effector proteins, we demonstrate that SopE dominantly triggers microvilli effacement and formation of membrane ruffles. Furthermore, SopE
Rac1 indirectly manipulates villin, which culminates in F-actin depolymerization. Collectively, these results indicate that SopE has dual functions during F-actin remodeling in PEC. While SopE-Rac1 triggers F-actin polymerization and ruffle formation, activation of PLCγ and villin by SopE depolymerizes F-actin in PEC. These results demonstrate the key role of SopE in destruction of the intestinal barrier during intestinal infection by
.
The invasion of polarized epithelial cells by Salmonella enterica requires the cooperative activity of the Salmonella pathogenicity island 1 (SPI1)-encoded type III secretion system (T3SS) and the ...SPI4-encoded adhesin SiiE. The invasion of polarized cells is more efficient than that of nonpolarized cells, and we observed the formation of clusters of bacteria on infected cells. Here we demonstrate that the invasion of polarized cells is a highly cooperative activity. Using a novel live-cell imaging approach, we visualized the cooperative entry of multiple bacteria into ruffles induced on the apical surfaces of polarized cells. The induction of membrane ruffles by activity of Salmonella enables otherwise noninvasive mutant strains to enter polarized host cells. Bacterial motility and chemotaxis were of lower importance for cooperativity in polarized-cell invasion. We propose that cooperative invasion is a key factor for the very efficient entry into polarized cells and a factor contributing to epithelial damage and intestinal inflammation.
Summary
Systemic administration of Salmonella enterica serovar Typhimurium to tumour bearing mice results in preferential colonization of the tumours and retardation of tumour growth. Although the ...bacteria are able to invade the tumour cells in vitro, in tumours they were never detected intracellularly. Ultrastructural analysis of Salmonella‐colonized tumours revealed that the bacteria had formed biofilms. Interestingly, depletion of neutrophilic granulocytes drastically reduced biofilm formation. Obviously, bacteria form biofilms in response to the immune reactions of the host. Importantly, we tested Salmonella mutants that were no longer able to form biofilms by deleting central regulators of biofilm formation. Such bacteria could be observed intracellularly in immune cells of the host or in tumour cells. Thus, tumour colonizing S. typhimurium might form biofilms as protection against phagocytosis. Since other bacteria are behaving similarly, solid murine tumours might represent a unique model to study biofilm formation in vivo.
(1) Background: the present study aimed to evaluate the effect of different types of warm-ups on the strength and skin temperature of Paralympic powerlifting athletes. (2) Methods: the participants ...were 15 male Paralympic powerlifting athletes. The effects of three different types of warm-up (without warm-up (WW), traditional warm-up (TW), or stretching warm-up (SW)) were analyzed on static and dynamic strength tests as well as in the skin temperature, which was monitored by thermal imaging. (3) Results: no differences in the dynamic and static indicators of the force were shown in relation to the different types of warm-ups. No significant differences were found in relation to peak torque (
= 0.055, F = 4.560, η2p = 0.246 medium effect), and one-repetition maximum (
= 0.139, F = 3.191, η2p = 0.186, medium effect) between the different types of warm-ups. In the thermographic analysis, there was a significant difference only in the pectoral muscle clavicular portion between the TW (33.04 ± 0.71 °C) and the WW (32.51 ± 0.74 °C) (
= 0.038). The TW method also presented slightly higher values than the SW and WW in the pectoral muscles sternal portion and the deltoid anterior portion, but with
-value > 0.05. (4) Conclusions: the types of warm-ups studied do not seem to interfere with the performance of Paralympic Powerlifting athletes. However, the thermal images showed that traditional warm-up best meets the objectives expected for this preparation phase.
Background
Inflammatory bowel diseases (IBD) are multifactorial disorders affecting millions of people worldwide with alarmingly increasing incidences every year. Dysfunction of the intestinal ...epithelial barrier is associated with IBD pathogenesis, and therapies include anti-inflammatory drugs that enhance intestinal barrier function. However, these drugs often have adverse side effects thus warranting the search for alternatives. Compatible solutes such as bacterial ectoines stabilize cell membranes and proteins.
Aim
To unravel whether ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and homoectoine (4,5,6,7-tetrahydro-2-methyl-1H-(1,3)-diazepine-4-carboxylic acid), a synthetic derivative of ectoine, have beneficial effects during dextran sulfate sodium (DSS)-induced colitis in mice.
Methods/Results
We found that the disease activity index was significantly reduced by both ectoines. DSS-induced edema formation, epithelial permeability, leukocyte recruitment and tissue damage were reduced by ectoine and homoectoine, with the latter having stronger effects. Interestingly, the claudin switch usually observed during colitis (decreased expression of claudin-1 and increased expression of the leaky claudin-2) was completely prevented by homoectoine, whereas ectoine only reduced claudin-2 expression. Concomitantly, only homoectoine ameliorated the drop in transepithelial electrical resistance induced by IFN-γ and TNF-α in Caco-2 cells. Both ectoines inhibited loss of ZO-1 and occludin and prevented IFN-γ/TNF-α-induced increased paracellular flux of 4 kDa FITC-dextran in vitro. Moreover, both ectoines reduced expression of pro-inflammatory cytokines and oxidative stress during colitis.
Conclusion
While both ectoine and homoectoine have protective effects on the epithelial barrier during inflammation, only homoectoine completely prevented the inflammatory claudin switch in tight junctions. Thus, homoectoine may serve as diet supplement in IBD patients to reach or extend remission.
Trimeric autotransporter adhesins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. TAAs form fibrous, adhesive structures on the bacterial cell surface. Their ...N-terminal extracellular domains are exported through a C-terminal membrane pore; the insertion of the pore domain into the bacterial outer membrane follows the rules of β-barrel transmembrane protein biogenesis and is dependent on the essential Bam complex. We have recently described the full fiber structure of SadA, a TAA of unknown function in Salmonella and other enterobacteria. In this work, we describe the structure and function of SadB, a small inner membrane lipoprotein. The sadB gene is located in an operon with sadA; orthologous operons are only found in enterobacteria, whereas other TAAs are not typically associated with lipoproteins. Strikingly, SadB is also a trimer, and its co-expression with SadA has a direct influence on SadA structural integrity. This is the first report of a specific export factor of a TAA, suggesting that at least in some cases TAA autotransport is assisted by additional periplasmic proteins.
Background: Autotransporter adhesins reach the bacterial cell surface by a complex mechanism.
Results: In the case of the autotransporter SadA from Salmonella, a lipoprotein assists in surface display.
Conclusion: The similarity to eukaryotic MATH domains suggests that the lipoprotein assists in trimerization of SadA.
Significance: Understanding the similarities between autotransport systems might lead to new ways of inhibiting bacterial adhesion.
Cancer is a major cause of death in children worldwide, with B-lineage cell acute lymphoblastic leukemia (B-ALL) being the most frequent childhood malignancy. Relapse, treatment failure and organ ...infiltration worsen the prognosis, warranting a better understanding of the implicated mechanisms. Cortactin is an actin-binding protein involved in cell adhesion and migration that is overexpressed in many solid tumors and in adult B-cell chronic lymphocytic leukemia. Here, we investigated cortactin expression and potential impact on infiltration and disease prognosis in childhood B-ALL. B-ALL cell lines and precursor cells from bone marrow (BM) and cerebrospinal fluid (CSF) of B-ALL patients indeed overexpressed cortactin. In CXCL12-induced transendothelial migration assays, transmigrated B-ALL cells had highest cortactin expression. In xenotransplantation models, only cortactin
-leukemic cells infiltrated lungs, brain, and testis; and they colonized more easily hypoxic BM organoids. Importantly, cortactin-depleted B-ALL cells were significantly less efficient in transendothelial migration, organ infiltration and BM colonization. Clinical data highlighted a significant correlation between high cortactin levels and BM relapse in drug-resistant high-risk B-ALL patients. Our results emphasize the importance of cortactin in B-ALL organ infiltration and BM relapse and its potential as diagnostic tool to identify high-risk patients and optimize their treatments.
Background: In Paralympic powerlifting (PP), athletes with and without spinal cord injury (SCI) compete in the same category. Athletes with SCI may be at a disadvantage in relation to the production ...of muscle strength and the execution of motor techniques. Objective: To analyze the indicators force, dynamic and static, at different intensities, on performance in athletes with and without SCI. Methods: The sample was composed of two groups of PP athletes: SCI (30.57 ± 4.20 years) and other deficiencies (OD; 25.67 ± 4.52 years). Athletes performed a test of maximum isometric force (MIF), time to MIF (Time), rate of force development (RFD), impulse, variability and fatigue index (FI), dynamic tests Mean Propulsive Velocity (MPV), Maximum Velocity (Vmax) and Power. Results: There were differences in the SCI in relation to OD, 50% 1RM (p < 0.05), in relation to MPV and Vmax. There were no differences in the static force indicators. Regarding EMG, there were differences between the SCI triceps in relation to the previous deltoid (p = 0.012). Conclusion: We concluded that the static and dynamic strength indicators are similar in Paralympic powerlifting athletes with spinal cord injury and other disabilities.
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