Ryland Young

Efficacy of Bacteriophage Therapy in a Model of Burkholderia cenocepacia Pulmonary Infection

The therapeutic potential of bacteriophages (phages) in a mouse model of acute Burkholderia cenocepacia pulmonary infection was assessed. Phage treatment was administered by either intranasal inhalation or intraperitoneal injection. Bacterial density, macrophage inflammatory protein 2 (MIP-2), and tumor necrosis factor alpha (TNF-alpha) levels were significantly reduced in lungs of mice treated with intraperitoneal phages (P < .05). No significant differences in lung bacterial density or MIP-2 levels were found between untreated mice and mice treated with intranasal phages, intraperitoneal ultraviolet-inactivated phages, or intraperitoneal lambda phage control mice. Mock-infected mice treated with phage showed no significant increase in lung MIP-2 or TNF-alpha levels compared with mock-infected/mock-treated mice. We have demonstrated the efficacy of phage therapy in an acute B. cenocepacia lung infection model. Systemic phage administration was more effective than inhalational administration, suggesting that circulating phages have better access to bacteria in lungs than do topical phages.

Phage lysis: three steps, three choices, one outcome.

The lysis of bacterial hosts by double-strand DNA bacteriophages, once thought to reflect merely the accumulation of sufficient lysozyme activity during the infection cycle, has been revealed to recently been revealed to be a carefully regulated and temporally scheduled process. For phages of Gramnegative hosts, there are three steps, corresponding to subversion of each of the three layers of the cell envelope: inner membrane, peptidoglycan, and outer membrane. The pathway is controlled at the level of the cytoplasmic membrane. In canonical lysis, a phage encoded protein, the holin, accumulates harmlessly in the cytoplasmic membrane until triggering at an allele-specific time to form micron-scale holes. This allows the soluble endolysin to escape from the cytoplasm to degrade the peptidoglycan. Recently a parallel pathway has been elucidated in which a different type of holin, the pinholin, which, instead of triggering to form large holes, triggers to form small, heptameric channels that serve to depolarize the membrane. Pinholins are associated with SAR endolysins, which accumulate in the periplasm as inactive, membrane-tethered enzymes. Pinholin triggering collapses the proton motive force, allowing the SAR endolysins to refold to an active form and attack the peptidoglycan. Surprisingly, a third step, the disruption of the outer membrane is also required. This is usually achieved by a spanin complex, consisting of a small outer membrane lipoprotein and an integral cytoplasmic membrane protein, designated as o-spanin and i-spanin, respectively. Without spanin function, lysis is blocked and progeny virions are trapped in dead spherical cells, suggesting that the outer membrane has considerable tensile strength. In addition to two-component spanins, there are some single-component spanins, or u-spanins, that have an N-terminal outer-membrane lipoprotein signal and a C-terminal transmembrane domain. A possible mechanism for spanin function to disrupt the outer membrane is to catalyze fusion of the inner and outer membranes.

The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes.

Bacteriophage λ has four adjacent genes –S, R, Rz and Rz1– dedicated to host cell lysis. While S, encoding the holin and antiholin, and R, encoding the endolysin, have been intensively studied, the products of Rz and Rz1 have not been characterized at either the structural or functional levels. Rz1 is an outer membrane lipoprotein and our results indicate that Rz is a type II signal anchor protein. Here we present evidence that an Rz–Rz1 complex that spans the periplasm carries out the final step in the process of host lysis. These results are discussed in terms of a model where endolysin‐mediated degradation of the cell wall is a prerequisite for conformational changes in the Rz–Rz1 complex leading to the juxtaposition and fusion of the IM and OM. Fusion of the two membranes removes the last physical barrier to efficient release of progeny virions.

Deletion analysis resolves cell-binding and lytic domains of the Pasteurella leukotoxin

A series of internal deletions in the IktA gene of Pasteurella haemofytica has been constructed. All of the deletions eliminated the lytic activity of the leukotoxin towards the bovine lymphoma cell line, BL‐3. Deletions removing segments of the amino‐proximal hydrophobic region, which is thought to constitute an essential membrane‐spanning domain, were found to agglutinate BL‐3 cells. Agglutination was similar to lysis by the wild‐type toxin in that it was dependent upon the presence of calcium and required expression of the lktC gene. The agglutinating deletion proteins protected BL‐3 cells from lysis by the wild‐type toxin in a competitive fashion. This suggests that these mutants bind to a surface feature of the leukocyte which interacts with the native leukotoxin. These findings demonstrate that the cell‐binding and lytic domains of the leukotoxin are separable.

Genomic and Functional Analyses of Rhodococcus equi Phages ReqiPepy6, ReqiPoco6, ReqiPine5, and ReqiDocB7

ABSTRACT The isolation and results of genomic and functional analyses of Rhodococcus equi phages ReqiPepy6, ReqiDocB7, ReqiPine5, and ReqiPoco6 (hereafter referred to as Pepy6, DocB7, Pine5, and Poco6, respectively) are reported. Two phages, Pepy6 and Poco6, more than 75% identical, exhibited genome organization and protein sequence likeness to Lactococcus lactis phage 1706 and clostridial prophage elements. An unusually high fraction, 27%, of Pepy6 and Poco6 proteins were predicted to possess at least one transmembrane domain, a value much higher than the average of 8.5% transmembrane domain-containing proteins determined from a data set of 36,324 phage protein entries. Genome organization and protein sequence comparisons place phage Pine5 as the first nonmycobacteriophage member of the large Rosebush cluster. DocB7, which had the broadest host range among the four isolates, was not closely related to any phage or prophage in the database, and only 23 of 105 predicted encoded proteins could be assigned a functional annotation. Because of the relationship of Rhodococcus to Mycobacterium, it was anticipated that these phages should exhibit some of the features characteristic of mycobacteriophages. Traits that were identified as shared by the Rhodococcus phages and mycobacteriophages include the prevalent long-tailed morphology and the presence of genes encoding LysB-like mycolate-hydrolyzing lysis proteins. Application of DocB7 lysates to soils amended with a host strain of R. equi reduced recoverable bacterial CFU, suggesting that phage may be useful in limiting R. equi load in the environment while foals are susceptible to infection.

Cultivation of lipid-producing bacteria with lignocellulosic biomass: Effects of inhibitory compounds of lignocellulosic hydrolysates

Lignocellulosic biomass has been recognized as a promising feedstock for the fermentative production of biofuel. However, the pretreatment of lignocellulose generates a number of by-products, such as furfural, 5-hydroxylmethyl furfural (5-HMF), vanillin, vanillic acids and trans-p-coumaric acid (TPCA), which are known to inhibit microbial growth. This research explores the ability of Rhodococcus opacus PD630 to use lignocellulosic biomass for production of triacylglycerols (TAGs), a common lipid raw material for biodiesel production. This study reports that R. opacus PD630 can grow well in R2A broth in the presence of these model inhibitory compounds while accumulating TAGs. Furthermore, strain PD630 can use TPCA, vanillic acid, and vanillin as carbon sources, but can only use TPCA and vanillic acid for TAG accumulation. Strain PD630 can also grow rapidly on the hydrolysates of corn stover, sorghum, and grass to accumulate TAGs, suggesting that strain PD630 is well-suited for bacterial lipid production from lignocellulosic biomass.

ESTIMATION OF DETECTION LIMITS OF THE PHAGE-INVASION BASED IDENTIFICATION OF BACTERIA

Recently a new method, SEnsing of Phage-Triggered Ion Cascade (SEPTIC) was proposed for the rapid detection and identification of bacteria via the electrical field caused by the stochastic emission of ions during phage infection. In this Letter, we present linear network theoretical considerations about the detection limits of the method. The considerations are based on our published data of the E. coli detection experiments and on the assumption of a linear response between the number of bacteria and the measured power density spectrum of the fluctuation-signal. Some practical limits of the detectability of the present agents with possible noise measurement arrangements are discussed in this paper. The calculations indicate that the detection and identification of a single bacterium can be achieved with natural (wild) phages with reasonable efforts within a time window of 10 minutes.

Genomic and proteomic features of mycobacteriophage SWU1 isolated from China soil

Mycobacteriophage SWU1 is a newly isolated phage from soil sample collected in Sichuan province, China using Mycobacterium smegmatis mc(2)155 as host. Plaque, phage morphology and one-step growth curve were characterized. The complete genomic sequence of phage SWU1 was determined by shotgun sequencing. The ends of SWU1 were determined. Structural proteins of SWU1 were analyzed by NanoLC-ESI-MS/MS. Seven ORFs were identified as structural protein encoded by SWU1 genome. The genetic basis underlying the SWU1 plaque was explored using comparative genomics. Prophages homologous to SWU1 were identified in two pathogens, Segniliparus rugosus ATCC BAA-974 and Mycobacterium rhodesiae JS60. Genus Segniliparus is a member of the order Corynebacteriales. To our knowledge, this is the first report of Mycobacterium prophages in different genera.

Inhibitory mechanism of the Qβ lysis protein A2

The lysis protein A2, present as a single copy on the surface of Qβ virion particles, was previously shown to inhibit the activity of MurA, an enzyme that catalyses the first committed step of murein biosynthesis. Here we report experiments with a two‐hybrid study that indicates A2 and MurA interact directly. Moreover, experiments with a soluble MBP–A2 fusion indicate that the interaction between MurA and A2 is dependent on a substrate‐induced conformational change featured in the UDP‐NAG‐liganded state of MurA but not the tetrahedral intermediate state. Moreover, based on the location of L138Q, the original dominant A2‐resistant mutant that identified MurA as the target, a directed mutagenesis strategy has identified a continuous surface required for A2 binding. This surface spans the catalytic loop/cleft and encompasses both the catalytic and C‐terminal domains. These data support a model in which A2 preferentially binds MurA liganded with UDP‐NAG, thereby preventing catalysis by occluding PEP from accessing the active site.

Cloning, sequencing and expression of a Pasteur el la haemolytica Al gene encoding a PurK-Iike protein

A membrane protein antigen of Pasteurella haemolytica Al encoded on the recombinant plasmid pYFC13 is isolated and characterized. Nucleotide sequence analysis of the insert DNA in pYFC13 identified the gene mpal which codes a protein of approximately 45 kDa without signal sequence. The deduced amino acids from the DNA sequence are homologous to Bacillus subtilis PurK by 29.4%; to Schizosaccharomycespombe Pur6 by 29.34%, to Saccharomyces cerevisiae Pur6 by 25.867%; and to 5. coli PurK by 25.223% identity, respectively. The purK and pur6 from these organisms are responsible for the activity of 5′-phosphoribosyl-5-amino-4-imidazole carboxylase which is involved in de novo purine biosynthesis. The protein was over-expressed in E. coli by its own promoter. The antigen we designated as Mpa1, could be localized to the cytoplasmic membrane of both P. haemolytica A1 and E. coli TB1 harbored pYFC13. The Mpa1 was antigenic in rabbit and in cattle since both animals produced antibody against this protein.