Explore the vast range of titles available.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B . pertussis , the causal agent of human whooping cough, and B . bronchiseptica , which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B . bronchiseptica , which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B . pertussis , which evolved from a B . bronchiseptica -like ancestor but now survives exclusively in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1 , ctaCDFGE1 , and cyoABCD1 . To test the hypothesis that the three cytochrome oxidases encoded within the B . pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B . bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo . No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B . pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B . bronchiseptica producing only the three B . pertussis -conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also determined that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on the environment at the surface of the ciliated epithelium, respiration requirements for bacteria that colonize the respiratory tract, and the evolution of virulence in bacterial pathogens.

Two-partner secretion (TPS) systems are broadly distributed among Gram-negative bacteria and play important roles in bacterial pathogenesis. FhaB-FhaC is the prototypical member of the TPS family and we here identified the protease that initiates a processing cascade that controls FhaB function. Filamentous hemagglutinin (FhaB) is a critical virulence factor for both Bordetella pertussis , the causal agent of whooping cough, and the closely related species Bordetella bronchiseptica . FhaB is an adhesin, suppresses inflammatory cytokine production, and protects against phagocytic cell clearance during infection. Regulated degradation of the FhaB C-terminal prodomain is required to establish a persistent infection in mice. Two proteases, CtpA in the periplasm and SphB1 on the bacterial surface, are known to mediate FhaB processing, and we recently determined that CtpA functions before, and controls the FhaB cleavage site of, SphB1. However, the data indicate that another periplasmic protease must initiate degradation of the prodomain by removing a portion of the FhaB C terminus that inhibits CtpA-mediated degradation. Using a candidate approach, we identified DegP as the initiating protease. Deletion of degP or substitution of its predicted catalytic residue resulted in reduced creation of FHA′ (the main product of FhaB processing) and an accumulation of full-length FhaB in whole-cell lysates. Also, FHA′ was no longer released into culture supernatants in degP mutants. Alterations of the FhaB C terminus that relieve inhibition of CtpA abrogate the need for DegP, consistent with DegP functioning prior to CtpA in the processing pathway. DegP is not required for secretion of FhaB through FhaC or for adherence of the bacteria to host cells, indicating that DegP acts primarily as a protease and not a chaperone for FhaB in B. bronchiseptica . Our results highlight a role for HtrA family proteases in activation of virulence factors in pathogenic bacteria. IMPORTANCE Two-partner secretion (TPS) systems are broadly distributed among Gram-negative bacteria and play important roles in bacterial pathogenesis. FhaB-FhaC is the prototypical member of the TPS family and we here identified the protease that initiates a processing cascade that controls FhaB function. Our results are significant because they provide insight into the molecular mechanism underlying the ability of Bordetella species to prevent clearance by phagocytic cells, which is critical for bacterial persistence in the lower respiratory tract. Our findings also highlight an underappreciated role for HtrA family proteases in processing specific bacterial virulence factors.

Bacterial arylmalonate decarboxylase (AMDase) shows high enantioselectivity and a broad substrate spectrum in the asymmetric synthesis of optically pure arylaliphatic carboxylic acids. The determination of the structure of AMDase has greatly extended the understanding of the catalytic mechanism of this unique cofactor-free decarboxylase and allowed the generation of tailor-made enzyme variants with improved catalytic properties. Despite this increase in knowledge and applicability, the natural role of the enzyme remains unknown. This mini-review summarizes the recent findings on the molecular mechanism and the synthetic application of the enzyme.

Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A–D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR–RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica.

To investigate the role played by urease during the Bordetella bronchiseptica infection process, the ability to colonise and persist in the mouse respiratory tract of a urease-negative B. bronchiseptica BB7865 and a BB7865 derivative constitutively expressing urease was compared with that of the wild-type strain. The results obtained showed that neither constitutive expression nor abolishment of urease activity had any significant effect on the course of B. bronchiseptica infection. Therefore, under our experimental conditions, urease is not essential for B. bronchiseptica to colonise and persist within the murine host.

Host-pathogen interactions are often driven by mechanisms that promote genetic variability. We have identified a group of temperate bacteriophages that generate diversity in a gene, designated mtd (major tropism determinant), which specifies tropism for receptor molecules on host Bordetella species. Tropism switching is the result of a template-dependent, reverse transcriptase-mediated process that introduces nucleotide substitutions at defined locations within mtd. This cassette-based mechanism is capable of providing a vast repertoire of potential ligand-receptor interactions.

In an effort to devise a safer and effective pertussis acelullar vaccine, outer membrane vesicles (OMVs) were engineered to decrease their endotoxicity. The pagL gene from Bordetella bronchiseptica, which encodes a lipid A 3-deacylase, was expressed in Bordetella pertussis strain Tohama I. The resulting OMVs, designated OMVsBpPagL, contain tetra- instead of penta-acylated LOS, in addition to pertussis surface immunogens such as pertactin and pertussis toxin, as the wild type OMVs. The characterized pertussis OMVsBpPagL were used in murine B. pertussis intranasal (i.n.) challenge model to examine their protective capacity when delivered by i.n. routes. Immunized BALB/c mice were challenged with sublethal doses of B. pertussis. Significant differences between immunized animals and the PBS treated group were observed (p<0.001). Adequate elimination rates (p<0.005) were observed in mice immunized either with OMVsBpPagL and wild type OMVs. All OMV preparations tested were non toxic according to WHO criteria; however, OMVsBpPagL displayed almost no weight loss at 3 days post administration, indicating less toxicity when compared with wild type OMVs. Induction of IL6- and IL1-expression in lung after i.n. delivery as well as neutrophil recruitment to airways showed coincident results, with a lower induction of the proinflammatory cytokines and lower recruitment in the case of OMVsBpPagL compared to wild type OMVs. Given their lower endotoxic activity and retained protective capacity in the mouse model, OMVsBpPagL obtained from B. pertussis seem as interesting candidates to be considered for the development of novel multi-antigen vaccine.

All publicly accessible microbial genome databases were searched for the occurrence of genes encoding proteins homologous to the cyanophycin synthetase (CphA) of Synechocystis sp. strain PCC 6803 in order to reveal the capability of microorganisms not belonging to the cyanobacteria to synthesize cyanophycin. Among 65 genome sequences, genes homologous to cphA were found in Acinetobacter sp. strain ADP1 (encoding a protein homologous to CphA with 40% amino acid identity), Bordetella bronchiseptica strain RB50 (39%), Bordetella pertussis strain Tohama I (39%), Bordetella parapertussis strain 12822 (39%), Clostridium botulinum strain ATCC 3502 (39%), Desulfitobacterium hafniense strain DCB-2 (38%) and Nitrosomonas europaea strain ATCC 25978 (37%). The gene homologous to cphA from Acinetobacter sp. strain DSM 587 was amplified by PCR, ligated to the vector pBluescript SK(-) downstream of the lac promoter and introduced into Escherichia coli. The recombinant strain of E. coli expressed CphA activity at up to 1.2 U/mg protein and accumulated cyanophycin to up to 7.5% of the cellular dry matter, indicating that CphA of Acinetobacter sp. strain DSM 587 is functionally active. In Acinetobacter sp. strain DSM 587 itself, cyanophycin accumulated to up to 1.4% of the total protein under phosphate-limited conditions, and cyanophycin synthetase activity was detected, which indicated the function of cyanophycin as a storage compound in this strain.

We have already isolated, purified, and characterized arylmalonate decarboxylases (AMDase; EC. 4.1.1.76) from Alcaligenes bronchisepticus KU1201 and Achromobacter sp. KU1311. These are unique enzymes that give optically pure α-arylpropionates from the corresponding α-aryl-α-methylmalonates. Recently, we have further screened novel AMDase producers from soil samples under acidic conditions and succeeded in isolating Enterobacter cloacae KU1313. The gene encoding the enzyme was cloned by polymerase chain reaction and sequenced. The AMDase gene consists of 720 nucleotides, which specifies a 240-amino-acid protein. The recombinant enzyme was purified and shown that the pH-activity profiles were quite different from those of known AMDases.

Previous studies suggested that the persistence in eukaryotic cells of a Bordetella bronchiseptica mutant carrying an insertion in the locus encoding the response regulator RisAS is impaired. This suggested that ris-dependent products are required for the intracellular survival of bacteria. In this study we demonstrate that ris-regulated products play a role in B. bronchiseptica resistance against both phagosomal acidification and reactive oxygen intermediates.