Calcium,Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.     

Streptococcus pneumoniae Phosphoglycerate Kinase Is a Novel Complement Inhibitor Affecting the Membrane Attack Complex Formation

The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that causes infections ranging from acute otitis media to life-threatening invasive disease. Pneumococci have evolved several strategies to circumvent the host immune response, in particular the complement attack. The pneumococcal glycolytic enzyme phosphoglycerate kinase (PGK) is both secreted and bound to the bacterial surface and simultaneously binds plasminogen and its tissue plasminogen activator tPA. In the present study we demonstrate that PGK has an additional role in modulating the complement attack. PGK interacted with the membrane attack complex (MAC) components C5, C7, and C9, thereby blocking the assembly and membrane insertion of MAC resulting in significant inhibition of the hemolytic activity of human serum. Recombinant PGK interacted in a dose-dependent manner with these terminal pathway proteins, and the interactions were ionic in nature. In addition, PGK inhibited C9 polymerization both in the fluid phase and on the surface of sheep erythrocytes. Interestingly, PGK bound several MAC proteins simultaneously. Although C5 and C7 had partially overlapping binding sites on PGK, C9 did not compete with either one for PGK binding. Moreover, PGK significantly inhibited MAC deposition via both the classical and alternative pathway at the pneumococcal surface. Additionally, upon activation plasmin(ogen) bound to PGK cleaved the central complement protein C3b thereby further modifying the complement attack. In conclusion, our data demonstrate for the first time to our knowledge a novel pneumococcal inhibitor of the terminal complement cascade aiding complement evasion by this important pathogen.     

Mutual Exclusivity of Hyaluronan and Hyaluronidase in Invasive Group A Streptococcus

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.     

Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC₅₀ of 0.9 μm at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.     

Structural Basis for Bacterial Quorum Sensing-mediated Oxalogenesis

The Burkholderia species utilize acetyl-CoA and oxaloacetate, substrates for citrate synthase in the TCA cycle, to produce oxalic acid in response to bacterial cell to cell communication, called quorum sensing. Quorum sensing-mediated oxalogenesis via a sequential reaction by ObcA and ObcB counteracts the population-collapsing alkaline pH of the stationary growth phase. Thus, the oxalic acid produced plays an essential role as an excreted public good for survival of the group. Here, we report structural and functional analyses of ObcA, revealing mechanistic features distinct from those of citrate synthase. ObcA exhibits a unique fold, in which a (β/α)₈-barrel fold is located in the C-domain with the N-domain inserted into a loop following α1 in the barrel fold. Structural analyses of the complexes with oxaloacetate and with a bisubstrate adduct indicate that each of the oxaloacetate and acetyl-CoA substrates is bound to an independent site near the metal coordination shell in the barrel fold. In catalysis, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr³²² as a general base, which then attacks the thioester carbonyl carbon of acetyl-CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal coordination shell and the absence of general acid(s) produces an unusual tetrahedral CoA adduct as a stable product. These results provide the structural basis for understanding the first step in oxalogenesis and constitute an example of the functional diversity of an enzyme for survival and adaptation in the environment.     

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.     

The basic keratin 10-binding domain of the virulence-associated pneumococcal serine-rich protein PsrP adopts a novel MSCRAMM fold

Streptococcus pneumoniae is a major human pathogen, and a leading cause of disease and death worldwide. Pneumococcal invasive disease is triggered by initial asymptomatic colonization of the human upper respiratory tract. The pneumococcal serine-rich repeat protein (PsrP) is a lung-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and promotes pneumococcal biofilm formation through self-oligomerization. We present the crystal structure of the KRT10-binding domain of PsrP (BR_187–385) determined to 2.0 Å resolution. BR_187–385 adopts a novel variant of the DEv-IgG fold, typical for microbial surface components recognizing adhesive matrix molecules adhesins, despite very low sequence identity. An extended β-sheet on one side of the compressed, two-sided barrel presents a basic groove that possibly binds to the acidic helical rod domain of KRT10. Our study also demonstrates the importance of the other side of the barrel, formed by extensive well-ordered loops and stabilized by short β-strands, for interaction with KRT10.     

Dose effects and density-dependent regulation of two microparasites of<Emphasis Type="Italic"> Daphnia magna</Emphasis>

Individual hosts constitute a limited resource for parasites, suggesting that density-dependent effects may play a role in within-host growth and parasite regulation. This hypothesis has been tested for several helminth parasites, but not for microparasites. We therefore examined dose-response patterns for the microparasitic bacterium Pasteuria ramosa and the fungus Metschnikowiella biscuspidata infecting the planktonic crustacean Daphnia magna. With increasing numbers of transmission stages administered to the host we found that host fecundity and survival and parasite transmission-stage production declined. Using a k-value analysis, a method that quantifies the strength of density dependence, we found for both parasites that density dependence acted at all doses, indicating the absence of a minimum density below which parasite fitness is density- independent. At low doses density was exactly compensated, but it was overcompensated at high doses. Overcompensation at high doses was weak for P. ramosa, but high for M. biscuspidata. At the two highest doses M. biscuspidata killed its hosts before any transmission stages were produced. Our data indicate that density dependence is expressed through retarded spore development in P. ramosa, but through both host mortality and reduced parasite fecundity in M. biscuspidata. A further experiment (P. ramosa only) revealed that in well-fed hosts more parasite transmission stages are produced than in poorly fed hosts, suggesting that competition for host resources retards P. ramosa development. Our data for P. ramosa, but not for M. biscuspidata, are largely consistent with assumptions made in models on microparasite epidemiology. We draw attention to the relevance of dose effects and within-host competition for the evolution of virulence. Received: 15 July 1999 / Accepted: 14 September 1999     

Two large-insert soybean genomic libraries constructed in a binary vector: applications in chromosome walking and genome wide physical mapping

Large DNA insert libraries in binary T-DNA vectors can assist in the isolation of the gene(s) underlying a quantitative trait locus (QTL). Binary vectors facilitate the transfer of large-insert DNA fragments containing a QTL from E. coli to Agrobacterium sp. and then to plants. We constructed two soybean large-insert libraries from cv. Forrest in the pCLD04541 (V41) binary vector after partial digestion of genomic high-molecular-weight DNA with BamHI or HindIII. The libraries contain 76,800 clones with an average insert size of 125 kb, and therefore represent 9.5-fold haploid genome equivalents. Colony hybridization using a chloroplast-specific probe infers that the libraries contain less than 0.5% clones of chloroplast DNA origin. These two libraries have provided clones for physical mapping of the soybean genome and for isolation of a number of disease resistance genes. One microsatellite marker was identified from the clone that hybridized to the Bng122 RFLP probe. The sequence-tagged site was used for genetic mapping and marker-assisted selection for genes underlying resistance to the soybean cyst nematode and sudden death syndrome. Received: 7 May 1999 / Accepted: 18 February 2000     

The development of larval resistance to a nucleopolyhedrovirus is not accompanied by an increased virulence in the virus

Two laboratory experiments were conducted to examine the possible coevolution of cabbage loopers (Trichoplusia ni) and their S nucleopolyhedrovirus (TnSNPV). At the conclusion of Experiments 1 and 2, T. ni had respectively evolved 4.4 × and 22 × resistance to TnSNPV. The higher level of resistance achieved in Experiment 2 could be due to marginally stronger selection, possibly greater genetic variability in larval resistance to TnSNPV, or both. However, the evolution of resistance was not accompanied by an increased virulence of TnSNPV or a change in the restriction profile of the viral DNA when digested with BamHI, EcoRI, HindIII, PstI, SalI, SstI or XhoI. Little genetic variability for virulence in the initial TnSNPV stocks, low mutation rates and possibly weak selection on the virus are some factors that may have constrained the evolution of TnSNPV. We discuss our results in light of the geographic mosaic theory of coevolution and their implications for the use of TnSNPV as a biological control agent against T. ni. This revised version was published online in November 2006 with corrections to the Cover Date.