Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.

Host immunity and cellular responses to bacterial outer membrane vesicles.

All Gram-negative bacteria produce outer membrane vesicles (OMVs) which are minute spherical structures emanating from the bacterial outer membrane. OMVs are primarily enriched in lipopolysaccharide (LPS) and phospholipids, as well as outer membrane and periplasmic proteins. Recent research has provided convincing evidence for their role in multiple aspects of bacterial physiology and their interaction with vertebrate host cells. OMVs play vital roles in bacterial colonization, delivery of virulence factors, and disease pathogenesis. Here, we discuss the interactions of OMVs with mammalian host cells with a focus on how bacteria use OMVs to modulate host immune responses that eventually enable bacteria to evade host immunity.

Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program.

It is generally believed that the last eukaryotic common ancestor (LECA) was a unicellular organism with motile cilia. In the vertebrates, the winged-helix transcription factor FoxJ1 functions as the master regulator of motile cilia biogenesis. Despite the antiquity of cilia, their highly conserved structure, and their mechanism of motility, the evolution of the transcriptional program controlling ciliogenesis has remained incompletely understood. In particular, it is presently not known how the generation of motile cilia is programmed outside of the vertebrates, and whether and to what extent the FoxJ1-dependent regulation is conserved. We have performed a survey of numerous eukaryotic genomes and discovered that genes homologous to foxJ1 are restricted only to organisms belonging to the unikont lineage. Using a mis-expression assay, we then obtained evidence of a conserved ability of FoxJ1 proteins from a number of diverse phyletic groups to activate the expression of a host of motile ciliary genes in zebrafish embryos. Conversely, we found that inactivation of a foxJ1 gene in Schmidtea mediterranea, a platyhelminth (flatworm) that utilizes motile cilia for locomotion, led to a profound disruption in the differentiation of motile cilia. Together, all of these findings provide the first evolutionary perspective into the transcriptional control of motile ciliogenesis and allow us to propose a conserved FoxJ1-regulated mechanism for motile cilia biogenesis back to the origin of the metazoans.

Acinetobacter baumannii: Virulence Strategies and Host Defense Mechanisms.

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen known to cause severe life-threatening infections, including pneumonia, meningitis, and sepsis. Recent emergence of this bacterium as a serious nosocomial pathogen has led to categorization of A. baumannii as a "high-priority" pathogen by the World Health Organization (WHO), for which research efforts are urgently required to develop therapeutic interventions. Some of the properties that make A. baumannii a serious pathogen include its capacity to tolerate high levels of stress and enhanced expression of efflux pumps that enable high degrees of antibiotic resistance. Virulence mechanisms employed by A. baumannii to establish successful infection and host responses elicited against A. baumannii to counter the infection are discussed in detail in this article.

Monitoring Mitochondrial Perturbations During Infection.

Mitochondria are pivotal organelles in the cell that regulate a myriad of cellular functions, which eventually govern cellular physiology and homeostasis. Intriguingly, microbial infection is known to trigger morphological and functional alterations of mitochondria. In fact, a number of bacteria and viruses have been reported to hijack mitochondrial functions including cell death induction and regulation of immune signaling to evade detection, promote their intracellular growth and subsequent dissemination. Here we describe methodologies that can be applied to assess mitochondrial functions upon infection. More specifically, we outline experimental procedures used to evaluate different parameters including mitochondrial morphology, adenosine triphosphate (ATP) levels, reactive oxygen species (ROS) levels, and mitophagy. Together these parameters can help gauge the overall health of mitochondria upon infection.

Acinetobacter baumannii Secretes a Bioactive Lipid That Triggers Inflammatory Signaling and Cell Death.

Acinetobacter baumannii is a highly pathogenic Gram-negative bacterium that causes severe infections with very high fatality rates. A. baumannii infection triggers innate as well as adaptive immunity, however, our understanding of the inflammatory factors secreted by A. baumannii that alarm the immune system remains limited. In this study, we report that the lab adapted and clinical strains of A. baumannii secrete an inflammatory bioactive factor which activates TLR2, leading to canonical IRAK4-dependent NF-κB signaling and production of pro-inflammatory cytokines interleukin (IL)-6 and IL-8 and activation of the inflammasome pathway causing pyroptotic cell death. Biochemical fractionation of the A. baumannii culture filtrate revealed the hydrophobic nature of the inflammatory factor. Concordantly, lipase treatment of the culture filtrate or TLR2 inhibition in macrophages abrogated NF-κB activation and cell death induction. Culture filtrates from the LPS- and lipoprotein-deficient A. baumannii mutants retain immuno-stimulatory properties suggesting that a lipid other than these known stimulatory molecules can trigger inflammation during A. baumannii infection. Our results reveal that A. baumannii secretes a previously unappreciated inflammatory bioactive lipid that activates multiple pro-inflammatory signaling pathways and induces cell death in human and murine macrophages.

Outer membrane vesicles containing OmpA induce mitochondrial fragmentation to promote pathogenesis of Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic resistant Gram-negative bacterium that causes life-threatening infections in humans with a very high mortality rate. A. baumannii is an extracellular pathogen with poorly understood virulence mechanisms. Here we report that A. baumannii employs the release of outer membrane vesicles (OMVs) containing the outer membrane protein A (OmpAAb) to promote bacterial pathogenesis and dissemination. OMVs containing OmpAAb are taken up by mammalian cells where they activate the host GTPase dynamin-related protein 1 (DRP1). OmpAAb mediated activation of DRP1 enhances its accumulation on mitochondria that causes mitochondrial fragmentation, elevation in reactive oxygen species (ROS) production and cell death. Loss of DRP1 rescues these phenotypes. Our data show that OmpAAb is sufficient to induce mitochondrial fragmentation and cytotoxicity since its expression in E. coli transfers its pathogenic properties to E. coli. A. baumannii infection in mice also induces mitochondrial damage in alveolar macrophages in an OmpAAb dependent manner. We finally show that OmpAAb is also required for systemic dissemination in the mouse lung infection model. In this study we uncover the mechanism of OmpAAb as a virulence factor in A. baumannii infections and further establish the host cell factor required for its pathogenic effects.

Mitochondrial Functions in Infection and Immunity.

Mitochondria have a central role in regulating a range of cellular activities and host responses upon bacterial infection. Multiple pathogens affect mitochondria dynamics and functions to influence their intracellular survival or evade host immunity. On the other side, major host responses elicited against infections are directly dependent on mitochondrial functions, thus placing mitochondria centrally in maintaining homeostasis upon infection. In this review, we summarize how different bacteria and viruses impact morphological and functional changes in host mitochondria and how this manipulation can influence microbial pathogenesis as well as the host cell metabolism and immune responses.

Nucleolar Function in Lifespan Regulation.

The nucleolus is a prominent membraneless organelle residing within the nucleus. The nucleolus has been regarded as a housekeeping structure mainly known for its role in ribosomal RNA (rRNA) production and ribosome assembly. However, accumulating evidence has revealed its functions in numerous cellular processes that control organismal physiology, thereby taking the nucleolus much beyond its conventional role in ribosome biogenesis. Perturbations in nucleolar functions have been associated with severe diseases such as cancer and progeria. Recent studies have also uncovered the role of the nucleolus in development and aging. In this review we discuss major nucleolar functions that impact organismal aging.

Nucleolar fibrillarin is an evolutionarily conserved regulator of bacterial pathogen resistance.

Innate immunity is the first line of defense against infections. Pathways regulating innate responses can also modulate other processes, including stress resistance and longevity. Increasing evidence suggests a role for the nucleolus in regulating cellular processes implicated in health and disease. Here we show the highly conserved nucleolar protein, fibrillarin, is a vital factor regulating pathogen resistance. Fibrillarin knockdown enhances resistance in C. elegans against bacterial pathogens, higher levels of fibrillarin induce susceptibility to infection. Pathogenic infection reduces nucleolar size, ribsosomal RNA, and fibrillarin levels. Genetic epistasis reveals fibrillarin functions independently of the major innate immunity mediators, suggesting novel mechanisms of pathogen resistance. Bacterial infection also reduces nucleolar size and fibrillarin levels in mammalian cells. Fibrillarin knockdown prior to infection increases intracellular bacterial clearance, reduces inflammation, and enhances cell survival. Collectively, these findings reveal an evolutionarily conserved role of fibrillarin in infection resistance and suggest the nucleolus as a focal point in innate immune responses.

Small nucleoli are a cellular hallmark of longevity.

Animal lifespan is regulated by conserved metabolic signalling pathways and specific transcription factors, but whether these pathways affect common downstream mechanisms remains largely elusive. Here we show that NCL-1/TRIM2/Brat tumour suppressor extends lifespan and limits nucleolar size in the major C. elegans longevity pathways, as part of a convergent mechanism focused on the nucleolus. Long-lived animals representing distinct longevity pathways exhibit small nucleoli, and decreased expression of rRNA, ribosomal proteins, and the nucleolar protein fibrillarin, dependent on NCL-1. Knockdown of fibrillarin also reduces nucleolar size and extends lifespan. Among wildtype C. elegans, individual nucleolar size varies, but is highly predictive for longevity. Long-lived dietary restricted fruit flies and insulin-like-peptide mutants exhibit small nucleoli and fibrillarin expression, as do long-lived dietary restricted and IRS1 knockout mice. Furthermore, human muscle biopsies from individuals who underwent modest dietary restriction coupled with exercise also display small nucleoli. We suggest that small nucleoli are a cellular hallmark of longevity and metabolic health conserved across taxa.