scholarly journals β1,2-Xylosyltransferase Cxt1p Is Solely Responsible for Xylose Incorporation into Cryptococcus neoformans Glycosphingolipids

2008 ◽  
Vol 7 (9) ◽  
pp. 1611-1615 ◽  
Author(s):  
Sherry A. Castle ◽  
Elizabeth A. Owuor ◽  
Stephanie H. Thompson ◽  
Michelle R. Garnsey ◽  
J. Stacey Klutts ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Structural Motif ◽  
Capsular Polysaccharides ◽  
Wild Type ◽  
Polysaccharide Biosynthesis ◽  
Mutant Strains

ABSTRACT The Manα1,3(Xylβ1,2)Manα structural motif is common to both capsular polysaccharides of Cryptococcus neoformans and to cryptococcal glycosphingolipids. Comparative analysis of glycosphingolipid structural profiles in wild-type and mutant strains showed that the Xylβ1,2-transferase (Cxt1p) that participates in capsular polysaccharide biosynthesis is also the sole transferase responsible for adding xylose to C. neoformans glycosphingolipids.

scholarly journals Parallel β-Helix Proteins Required for Accurate Capsule Polysaccharide Synthesis and Virulence in the Yeast Cryptococcus neoformans

2007 ◽  
Vol 6 (4) ◽  
pp. 630-640 ◽  
Author(s):  
Oliver W. Liu ◽  
Mark J. S. Kelly ◽  
Eric D. Chow ◽  
Hiten D. Madhani
Keyword(s):  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Resonance Spectroscopy ◽  
Wild Type ◽  
Repeat Proteins ◽  
Knockout Mutants ◽  
Polysaccharide Synthesis ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Opportunistic Fungal Pathogen

ABSTRACT The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an α-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel β-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.

scholarly journals Polysaccharides and virulence of Burkholderia pseudomallei

2007 ◽  
Vol 56 (8) ◽  
pp. 1005-1010 ◽  
Author(s):  
M. Sarkar-Tyson ◽  
J. E. Thwaite ◽  
S. V. Harding ◽  
S. J. Smither ◽  
P. C. F. Oyston ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Capsular Polysaccharide ◽  
Gene Clusters ◽  
Type I ◽  
Wild Type ◽  
Time To Death ◽  
Type Iii ◽  
Type Iv ◽  
Mutant Strains ◽  
Delayed Time

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease of humans and animals. Gene clusters which encode capsular polysaccharide (type I O-PS) and LPS (type II O-PS), both of which play roles in virulence, have previously been identified. Here, the identification of two further putative clusters, type III O-PS and type IV O-PS, is reported. Mice challenged with type III O-PS or type IV O-PS mutants showed increased mean times to death (7.8 and 11.6 days) compared to those challenged with wild-type B. pseudomallei (3 days). To investigate the possible roles of polysaccharides in protection, mice were immunized with killed cells of wild-type B. pseudomallei or killed cells of B. pseudomallei with mutations in the O antigen, capsular polysaccharide, type III O-PS or type IV O-PS gene clusters. Immunization with all polysaccharide mutant strains resulted in delayed time to death compared to the naïve controls, following challenge with wild-type B. pseudomallei strain K96243. However, immunization with killed polysaccharide mutant strains conferred different degrees of protection, demonstrating the immunological importance of the polysaccharide clusters on the surface of B. pseudomallei.

scholarly journals Envelope Proteins of the CsmB/CsmF and CsmC/CsmD Motif Families Influence the Size, Shape, and Composition of Chlorosomes in Chlorobaculum tepidum

2009 ◽  
Vol 191 (22) ◽  
pp. 7109-7120 ◽  
Author(s):  
Hui Li ◽  
Donald A. Bryant
Keyword(s):  
Size Composition ◽  
Envelope Proteins ◽  
Structural Motif ◽  
Supramolecular Organization ◽  
Wild Type ◽  
Absorbance Maximum ◽  
Chlorobaculum Tepidum ◽  
Mutant Strains ◽  
Redundant Functions ◽  
Mutational Study

ABSTRACT The chlorosome envelope of Chlorobaculum tepidum contains 10 proteins that belong to four structural motif families. A previous mutational study (N.-U. Frigaard, H. Li, K. J. Milks, and D. A. Bryant, J. Bacteriol. 186:646-653, 2004) suggested that some of these proteins might have redundant functions. Six multilocus mutants were constructed to test the effects of eliminating the proteins of the CsmC/CsmD and CsmB/CsmF motif families, and the resulting strains were characterized physiologically and biochemically. Mutants lacking all proteins of either motif family still assembled functional chlorosomes, and as measured by growth rates of the mutant strains, light harvesting was affected only at the lowest light intensities tested (9 and 32 μmol photons m−2 s−1). The size, composition, and biogenesis of the mutant chlorosomes differed from those of wild-type chlorosomes. Mutants lacking proteins of the CsmC/CsmD motif family produced smaller chlorosomes than did the wild type, and the Qy absorbance maximum for the bacteriochlorophyll c aggregates in these chlorosomes was strongly blueshifted. Conversely, the chlorosomes of mutants lacking proteins of the CsmB/CsmF motif family were larger than wild-type chlorosomes, and the Qy absorption for their bacteriochlorophyll c aggregates was redshifted. When CsmH was eliminated in addition to other proteins of either motif family, chlorosomes had smaller diameters. These data show that the chlorosome envelope proteins of the CsmB/CsmF and CsmC/CsmD families play important roles in determining chlorosome size as well as the assembly and supramolecular organization of the bacteriochlorophyll c aggregates within the chlorosome.

scholarly journals Francisella tularensisSchu S4 O-Antigen and Capsule Biosynthesis Gene Mutants Induce Early Cell Death in Human Macrophages

2010 ◽  
Vol 79 (2) ◽  
pp. 581-594 ◽  
Author(s):  
Stephen R. Lindemann ◽  
Kaitian Peng ◽  
Matthew E. Long ◽  
Jason R. Hunt ◽  
Michael A. Apicella ◽  
...  
Keyword(s):  
Cell Death ◽  
High Throughput Screening ◽  
Capsular Polysaccharide ◽  
Virulent Strain ◽  
Wild Type ◽  
Intracellular Growth ◽  
Human Macrophages ◽  
Polysaccharide Biosynthesis ◽  
O Antigen ◽  
Schu S4

ABSTRACTFrancisella tularensisis capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains ofFrancisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of theFrancisellachromosome,FTT1236,FTT1237, andFTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest thatFTT1236,FTT1237, andFTT1238are important for polysaccharide biosynthesis and that theFrancisellaO antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.

scholarly journals Genetic Diversity of the Capsular Polysaccharide C Biosynthesis Region of Bacteroides fragilis

2000 ◽  
Vol 68 (11) ◽  
pp. 6182-6188 ◽  
Author(s):  
Laurie E. Comstock ◽  
Annalisa Pantosti ◽  
Dennis L. Kasper
Keyword(s):  
Genetic Diversity ◽  
Capsular Polysaccharide ◽  
Bacteroides Fragilis ◽  
Hybridization Analysis ◽  
Clinical Isolates ◽  
Pcr Analysis ◽  
Capsular Polysaccharides ◽  
Genetic Approach ◽  
Polysaccharide Biosynthesis ◽  
Putative Aminotransferase

ABSTRACT A genetic approach was used to assess the heterogeneity of the capsular polysaccharide C (PS C) biosynthesis locus ofBacteroides fragilis and to determine whether distinct loci contain genes whose products are likely to be involved in conferring charged groups that enable the B. fragilis capsular polysaccharides to induce abscesses. A collection of 50 B. fragilis strains was examined. PCR analysis demonstrated that the genes flanking the PS C biosynthesis region are conserved, whereas the genes within the loci are heterogeneous. OnlycfiA + B. fragilis strains, which represent 3% of the clinical isolates of B. fragilis, displayed heterogeneity in the regions flanking the polysaccharide biosynthesis genes. Primers were designed in the conserved regions upstream and downstream of the PS C locus and were used to amplify the region from 45 of the 50 B. fragilis strains studied. Fourteen PS C genetic loci could be differentiated by a combination of PCR and extended PCR. These loci ranged in size from 14 to 26 kb. Hybridization analysis with genes from the PS C loci of strains 9343 and 638R revealed that the majority of strains contain homologs ofwcgC (N-acetylmannosamine dehydrogenase),wcfF (putative dehydrogenase), and wcgP(putative aminotransferase). The data suggest that the synthesis of polysaccharides that have zwitterionic characteristics rendering them able to induce abscesses is common in B. fragilis.

scholarly journals Involvement of CD14, Toll-Like Receptors 2 and 4, and MyD88 in the Host Response to the Fungal Pathogen Cryptococcus neoformans In Vivo

2004 ◽  
Vol 72 (9) ◽  
pp. 5373-5382 ◽  
Author(s):  
Lauren E. Yauch ◽  
Michael K. Mansour ◽  
Shmuel Shoham ◽  
James B. Rottman ◽  
Stuart M. Levitz
Keyword(s):  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Adaptor Protein ◽  
Interleukin 4 ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Histopathologic Analysis ◽  
And Control

ABSTRACT The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88−/− mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14−/− mice had reduced survival when infected i.v., while TLR2−/− mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14−/−, TLR2−/−, and MyD88−/− mice. MyD88−/− mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88−/− mice correlating with increased numbers of lung CFU and serum and lung GXM levels.

scholarly journals Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 591-604 ◽  
Author(s):  
Kaihei Kojima ◽  
Yong-Sun Bahn ◽  
Joseph Heitman
Keyword(s):  
Drug Resistance ◽  
Cryptococcus Neoformans ◽  
Type Strain ◽  
Wild Type Strain ◽  
Mapk Pathway ◽  
Control Plant ◽  
Antifungal Drug ◽  
Cell Swelling ◽  
Wild Type ◽  
Mutant Strains

Fludioxonil is employed as an agricultural fungicide to control plant-pathogenic fungi such as Botrytis cinerea. Cryptococcus neoformans is a basidiomycetous human fungal pathogen that causes fatal disease in immunocompromised hosts. This paper demonstrates that three different signalling cascades regulate sensitivity of C. neoformans to fludioxonil. Fludioxonil inhibited growth of the serotype A sequence reference strain H99 but not that of the sequenced serotype D strain JEC21. In the drug-sensitive wild-type strain, fludioxonil exposure activated the Hog1 osmosensing pathway, and hog1Δ mutations conferred fludioxonil resistance. Fludioxonil treatment caused cell growth inhibition following cell swelling and cytokinesis defects in the sensitive wild-type but not in a hog1Δ mutant strain, suggesting that Hog1 activation results in morphological cellular defects. Fludioxonil exerted a fungistatic effect on the wild-type strain H99, but exhibited fungicidal activity against calcineurin mutant strains, indicating that the calcineurin pathway contributes to drug resistance in this fungus. Combination of fludioxonil and the calcineurin inhibitor FK506 synergistically inhibited C. neoformans growth. mpk1Δ MAPK mutant strains exhibited fludioxonil hypersensitivity, indicating that this pathway also contributes to drug resistance. These studies provide evidence that the broad-spectrum antifungal drug fludioxonil exerts its action via activation of the Hog1 MAPK pathway and provide insight into novel targets for synergistic antifungal drug combinations.

scholarly journals Analysis of a Capsular Polysaccharide Biosynthesis Locus of Bacteroides fragilis

1999 ◽  
Vol 67 (7) ◽  
pp. 3525-3532 ◽  
Author(s):  
Laurie E. Comstock ◽  
Michael J. Coyne ◽  
Arthur O. Tzianabos ◽  
Annalisa Pantosti ◽  
Andrew B. Onderdonk ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Bacteroides Fragilis ◽  
Open Reading Frames ◽  
Capsular Polysaccharides ◽  
Nucleotide Sugar ◽  
Polysaccharide Biosynthesis ◽  
Abdominal Abscesses ◽  
Intra Abdominal Infection ◽  
Reading Frames ◽  
Nucleotide Sugar Biosynthesis

ABSTRACT A major clinical manifestation of infection with Bacteroides fragilis is the formation of intra-abdominal abscesses, which are induced by the capsular polysaccharides of this organism. Transposon mutagenesis was used to locate genes involved in the synthesis of capsular polysaccharides. A 24,454-bp region was sequenced and found to contain a 15,379-bp locus (designated wcf) with 16 open reading frames (ORFs) encoding products similar to those encoded by genes of other bacterial polysaccharide biosynthesis loci. Four genes encode products that are similar to enzymes involved in nucleotide sugar biosynthesis. Seven genes encode products that are similar to sugar transferases. Two gene products are similar toO-acetyltransferases, and two products are probably involved in polysaccharide transport and polymerization. The product of one ORF, WcfH, is similar to a set of deacetylases of the NodB family. Deletion mutants demonstrated that the wcf locus is necessary for the synthesis of polysaccharide B, one of the two capsular polysaccharides of B. fragilis 9343. The virulence of the polysaccharide B-deficient mutant was comparable to that of the wild type in terms of its ability to induce abscesses in a rat model of intra-abdominal infection.

scholarly journals The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 4058-4068 ◽  
Author(s):  
Anat Lerner ◽  
Susana Castro-Sowinski ◽  
Angel Valverde ◽  
Hadas Lerner ◽  
Rachel Dror ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Plant Root ◽  
Extracellular Polysaccharide ◽  
Extracellular Polysaccharides ◽  
Wild Type ◽  
Polysaccharide Biosynthesis ◽  
Beneficial Effects ◽  
Mutant Strains ◽  
Azospirillum Brasilense Sp7 ◽  
Surface Polysaccharides

Azospirillum brasilense is a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid of A. brasilense Sp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes, noeJ and noeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that in A. brasilense Sp7, noeJ and noeL are involved in lipopolysaccharide and exopolysaccharide synthesis. noeJ and noeL mutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, both noeJ and noeL mutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption of noeL, but not noeJ, affected the ability of the A. brasilense Sp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.

scholarly journals Adenylyl Cyclase Functions Downstream of the Gα Protein Gpa1 and Controls Mating and Pathogenicity of Cryptococcus neoformans

2002 ◽  
Vol 1 (1) ◽  
pp. 75-84 ◽  
Author(s):  
J. Andrew Alspaugh ◽  
Read Pukkila-Worley ◽  
Toshiaki Harashima ◽  
Lora M. Cavallo ◽  
Deanna Funnell ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Signal Transduction Pathway ◽  
Growth Defect ◽  
Wild Type ◽  
Camp Production ◽  
Mutant Strains ◽  
Opportunistic Fungal Pathogen ◽  
Mutant Phenotypes

ABSTRACT The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Gα subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Gα protein Gpa1, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpa1 mutant strains. Physiological studies revealed that the Gα protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

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