A Pseudo-tRNA Modulates Antibiotic Resistance in \u3cem\u3eBacillus cereus\u3c/em\u3e

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNAOther, which does not function in translation. Deletion of tRNAOther led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNAOther. This suggested that tRNAOther might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3′ and 5′-ends of tRNAOther. tRNAOther expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5′ leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNAOther may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell

A Pseudo-tRNA Modulates Antibiotic Resistance in Bacillus cereus

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNA(Other), which does not function in translation. Deletion of tRNA(Other) led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNA(Other). This suggested that tRNA(Other) might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 39 and 5\u27-ends of tRNA(Other). tRNA(Other) expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 59 leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNA(Other) may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell

Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI

The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C(+) (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C(+) macrophages and Ly6C(-) macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI-and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation

The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell

High-Throughput Microplate-Based Assay to Monitor Plasma Membrane Wounding and Repair

The plasma membrane of mammalian cells is susceptible to disruption by mechanical and biochemical damages that frequently occur within tissues. Therefore, efficient and rapid repair of the plasma membrane is essential for maintaining cellular homeostasis and survival. Excessive damage of the plasma membrane and defects in its repair are associated with pathological conditions such as infections, muscular dystrophy, heart failure, diabetes, and lung and neurodegenerative diseases. The molecular events that remodel the plasma membrane during its repair remain poorly understood. In the present work, we report the development of a quantitative high-throughput assay that monitors the efficiency of the plasma membrane repair in real time using a sensitive microplate reader. In this assay, the plasma membrane of living cells is perforated by the bacterial pore-forming toxin listeriolysin O and the integrity and recovery of the membrane are monitored at 37°C by measuring the fluorescence intensity of the membrane impermeant dye propidium iodide. We demonstrate that listeriolysin O causes dose-dependent plasma membrane wounding and activation of the cell repair machinery. This assay was successfully applied to cell types from different origins including epithelial and muscle cells. In conclusion, this high-throughput assay provides a novel opportunity for the discovery of membrane repair effectors and the development of new therapeutic compounds that could target membrane repair in various pathological processes, from degenerative to infectious diseases

LysPGS Formation in Listeria monocytogenes Has Broad Roles in Maintaining Membrane Integrity Beyond Antimicrobial Peptide Resistance

Listeria monocytogenes is an intracellular, foodborne gastrointestinal pathogen that is primarily responsible for causing listeriosis or food poisoning in otherwise healthy individuals. Infections that arise during pregnancy or within immune compromised individuals are much more serious resulting in the risk of fetal termination or fetal fatality postpartum in the former and septicemia or meningitis with a 20% fatality rate in the latter. While the roles of internalin proteins and listeriolysin-O in the infection process are well characterized, the specific roles of lysine-modified phospholipids in the membrane of L. monocytogenes are not. Investigation into the lipid bilayer composition of L. monocytogenes indicated that the overall proportions of lipids, including lysylcardiolipin and lysylphosphatidylglycerol (LysPG), vary with growth temperature and growth phase. In addition, we demonstrate that LysPG formation is essential for L. monocytogenes survival in the presence of increased osmolytic stress but has no effect on bacterial adherence, invasion or survival in the presence of physiologically relevant concentrations of human neutrophil peptide (HNP-1). In the absence of LysPG synthesis, L. monocytogenes unexpectedly retained flagellum-mediated motility at 37 °C. Taken together, these findings show that LysPG formation in L. monocytogenes has broader functions in virulence and survival beyond its known role in the modification of membrane potential previously observed in other bacteria

Yersinia enterocolitica Inhibits Salmonella enterica Serovar Typhimurium and Listeria monocytogenes Cellular Uptake

Yersinia enterocolitica biovar 1B employs two type three secretion systems (T3SS), Ysa and Ysc, which inject effector proteins into macrophages to prevent phagocytosis. Conversely, Salmonella enterica serovar Typhimurium uses a T3SS encoded by Salmonella pathogenicity island 1 (SPI1) to actively invade cells that are normally nonphagocytic and a second T3SS encoded by SPI2 to survive within macrophages. Given the distinctly different outcomes that occur with regard to host cell uptake of S. Typhimurium and Y. enterocolitica, we investigated how each pathogen influences the internalization outcome of the other. Y. enterocolitica reduces S. Typhimurium invasion of HeLa and Caco-2 cells to a level similar to that observed using an S. Typhimurium SPI1 mutant alone. However, Y. enterocolitica had no effect on S. Typhimurium uptake by J774.1 or RAW264.7 macrophage-like cells. Y. enterocolitica was also able to inhibit the invasion of epithelial and macrophage-like cells by Listeria monocytogenes. Y. enterocolitica mutants lacking either the Ysa or Ysc T3SS were partially defective, while double mutants were completely defective, in blocking S. Typhimurium uptake by epithelial cells. S. Typhimurium encodes a LuxR homolog, SdiA, which detects N-acylhomoserine lactones (AHLs) produced by Y. enterocolitica and upregulates the expression of an invasin (Rck) and a putative T3SS effector (SrgE). Two different methods of constitutively activating the S. Typhimurium SdiA regulon failed to reverse the uptake blockade imposed by Y. enterocolitica

A Pseudo-tRNA Modulates Antibiotic Resistance in Bacillus cereus

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNA(Other), which does not function in translation. Deletion of tRNA(Other) led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNA(Other). This suggested that tRNA(Other) might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3' and 5'-ends of tRNA(Other). tRNA(Other) expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5' leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNA(Other) may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell