During cell stress, Saccharomyces cerevisiae increases the synthesis of chitin and glucans to strengthen and repair the cell wall. In this study, we show that under conditions of cell stress, the ...steady-state localization of chitin synthase III (Chs3p) shifts from internal stores (chitosomes) to the plasma membrane (PM). This redistribution occurs rapidly and requires the activators of the cell wall stress response signaling pathway, the G protein Rho1p, and the protein kinase Pkc1p, but not the cell integrity response mitogen-activated protein kinase cascade. Furthermore, expression of activated forms of Rho1p or Pkc1p, in the absence of cell stress, is sufficient to redistribute Chs3p to the PM. In cells deficient for both the clathrin adaptor complex 1 and Chs6p, where Chs3p is transported to the PM by an alternative bypass pathway, cell wall stress did not cause mobilization of Chs3p, suggesting that Rho1p/Pkc1p regulate Chs3p exit from the trans-Golgi network. The mobilization of an intracellular reservoir of Chs3p presents a novel opportunity to investigate the genetic basis of regulated vesicular traffic.
Summary
Chlamydia sp. are responsible for a wide range of diseases of significant clinical and public health importance. In this review, we highlight how recent cellular and functional genomic ...approaches have significantly increased our knowledge of the pathogenic mechanisms used by these genetically intractable bacteria. As the extensive repertoire of chlamydial proteins that are translocated into the mammalian host is identified and characterized, a molecular understanding of how Chlamydiae co‐opt host cellular functions and block innate immune pathways is beginning to emerge.
A major roadblock to understanding how microbes in the gastrointestinal tract colonize and influence the physiology of their hosts is our inability to genetically manipulate new bacterial species and ...experimentally assess the function of their genes. We describe the application of population-based genomic sequencing after chemical mutagenesis to map bacterial genes responsible for motility in Exiguobacterium acetylicum, a representative intestinal Firmicutes bacterium that is intractable to molecular genetic manipulation. We derived strong associations between mutations in 57 E. acetylicum genes and impaired motility. Surprisingly, less than half of these genes were annotated as motility-related based on sequence homologies. We confirmed the genetic link between individual mutations and loss of motility for several of these genes by performing a large-scale analysis of spontaneous suppressor mutations. In the process, we reannotated genes belonging to a broad family of diguanylate cyclases and phosphodiesterases to highlight their specific role in motility and assigned functions to uncharacterized genes. Furthermore, we generated isogenic strains that allowed us to establish that Exiguobacterium motility is important for the colonization of its vertebrate host. These results indicate that genetic dissection of a complex trait, functional annotation of new genes, and the generation of mutant strains to define the role of genes in complex environments can be accomplished in bacteria without the development of species-specific molecular genetic tools.
Pathogenic bacteria are armed with potent effector proteins that subvert host signalling processes during infection
. The activities of bacterial effectors and their associated roles within the host ...cell are often poorly understood, particularly for Chlamydia trachomatis
, a World Health Organization designated neglected disease pathogen. We identify and explain remarkable dual Lys63-deubiquitinase (DUB) and Lys-acetyltransferase activities in the Chlamydia effector ChlaDUB1. Crystal structures capturing intermediate stages of each reaction reveal how the same catalytic centre of ChlaDUB1 can facilitate such distinct processes, and enable the generation of mutations that uncouple the two activities. Targeted Chlamydia mutant strains allow us to link the DUB activity of ChlaDUB1 and the related, dedicated DUB ChlaDUB2 to fragmentation of the host Golgi apparatus, a key process in Chlamydia infection for which effectors have remained elusive. Our work illustrates the incredible versatility of bacterial effector proteins, and provides important insights towards understanding Chlamydia pathogenesis.
The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii ...replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpD and ΔcvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ΔcvpB and ΔcvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.
The obligate intracellular pathogen Chlamydia trachomatis secretes effector proteins across the membrane of the pathogen-containing vacuole (inclusion) to modulate host cellular functions. In an ...immunological screen for secreted chlamydial proteins, we identified CT049 and CT050 as potential inclusion membrane-associated proteins. These acidic, nonglobular proteins are paralogously related to the passenger domain of the polymorphic membrane protein PmpC and, like other Pmp proteins, are highly polymorphic among C. trachomatis ocular and urogenital strains. We generated antibodies to these Pmp-like secreted (Pls) proteins and determined by immunofluorescence microscopy that Pls1 (CT049) and Pls2 (CT050) localized to globular structures within the inclusion lumen and at the inclusion membrane. Fractionation of membranes and cytoplasmic components from infected cells by differential and density gradient centrifugation further indicated that Pls1 and Pls2 associated with membranes distinct from the bulk of bacterial and inclusion membranes. The accumulation of Pls1 and, to a lesser extent, Pls2 in the inclusion lumen was insensitive to the type III secretion inhibitor C1, suggesting that this translocation system is not essential for Pls protein secretion. In contrast, Pls secretion and stability were sensitive to low levels of β-lactam antibiotics, suggesting that a functional cell wall is required for Pls secretion from the bacterial cell. Finally, we tested the requirement for these proteins in Chlamydia infection by microinjecting anti-Pls1 and anti-Pls2 antibodies into infected cells. Coinjection of anti-Pls1 and -Pls2 antibodies partially inhibited expansion of the inclusion. Because Pls proteins lack classical sec-dependent secretion signals, we propose that Pls proteins are secreted into the inclusion lumen by a novel mechanism to regulate events important for chlamydial replication and inclusion expansion.
Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (
= 119), ...the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of
, a species of mucin-degrading bacteria (
= 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded
and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded
and thinned the colonic mucus layer. Antibiotic treatment to eradicate
before caloric restriction preserved colonic mucus, whereas
reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing
in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting
or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.
Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, includingChlamydia trachomatis Although nearly all of the enzymes in ...the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogenC. trachomatislacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against aC. trachomatisgenomic library using a conditional-lethallpxHmutantEscherichia colistrain, we have identified an open reading frame (Ct461, renamedlpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function inE. coliand catalyzes the conversion of UDP-DAGn to lipid Xin vitro LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity ofC. trachomatis, validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools forC. trachomatisand should be broadly applicable for the functional characterization of other essentialC. trachomatisgenes.IMPORTANCEChlamydia trachomatisis a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of manyChlamydiagenes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against theC. trachomatisgenomic library using a conditional-lethal mutant ofE. coliand identified the missing essential gene in the lipid A biosynthetic pathway, which we designatedlpxG We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activityin vitro Overexpression of LpxG inC. trachomatisleads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating thein vivofunction of LpxG and highlighting the importance of regulated lipid A biosynthesis inC. trachomatis.
Objective
The purpose of this study was to establish a biorepository of clinical, metabolomic, and microbiome samples from adolescents with obesity as they undergo lifestyle modification.
Methods
A ...total of 223 adolescents aged 10 to 18 years with BMI ≥95th percentile were enrolled, along with 71 healthy weight participants. Clinical data, fasting serum, and fecal samples were collected at repeated intervals over 6 months. Herein, the study design, data collection methods, and interim analysis—including targeted serum metabolite measurements and fecal 16S ribosomal RNA gene amplicon sequencing among adolescents with obesity (n = 27) and healthy weight controls (n = 27)—are presented.
Results
Adolescents with obesity have higher serum alanine aminotransferase, C‐reactive protein, and glycated hemoglobin, and they have lower high‐density lipoprotein cholesterol when compared with healthy weight controls. Metabolomics revealed differences in branched‐chain amino acid–related metabolites. Also observed was a differential abundance of specific microbial taxa and lower species diversity among adolescents with obesity when compared with the healthy weight group.
Conclusions
The Pediatric Metabolism and Microbiome Study (POMMS) biorepository is available as a shared resource. Early findings suggest evidence of a metabolic signature of obesity unique to adolescents, along with confirmation of previously reported findings that describe metabolic and microbiome markers of obesity.
Abstract
The secreted Chlamydia protease CPAF cleaves a defined set of mammalian and Chlamydia proteins in vitro. As a result, this protease has been proposed to modulate a range of bacterial and ...host cellular functions. However, it has recently come into question the extent to which many of its identified substrates constitute bona fide targets of proteolysis in infected host cell rather than artifacts of postlysis degradation. Here, we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. Using reverse genetic approaches, we identified two C. trachomatis strains possessing nonsense, loss of function mutations in cpa (CT858) and a third strain containing a mutation in type II secretion (T2S) machinery that inhibited CPAF activity by blocking zymogen secretion and subsequent proteolytic maturation into the active hydrolase. HeLa cells infected with T2S− or CPAF−C. trachomatis mutants lacked detectable in vitro CPAF proteolytic activity and were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine induced apoptosis, Golgi fragmentation, altered NFκB dependent gene expression, and resistance to reinfection. However, CPAF deficient mutants did display impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C. trachomatis. In addition, we provide compelling evidence in live cells that CPAF mediated protein processing of at least two host protein targets, vimentin filaments and the nuclear envelope protein lamin associated protein 1 (LAP1), occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. CPAF dependent processing of host proteins correlates with a loss of inclusion membrane integrity, and so we propose that CPAF plays a role late in infection, possibly during the stages leading to the dismantling of the infected cell prior to the release of EBs during cell lysis.
The identification of mutants in a key virulence protease in Chlamydia helps clarify the role that this protease plays in manipulating host cellular functions.
The identification of mutants in a key virulence protease in Chlamydia helps clarify the role that this protease plays in manipulating host cellular functions.
PDF
