Showing papers by "Richard W. Titball published in 2010"

A genomic survey of positive selection in Burkholderia pseudomallei provides insights into the evolution of accidental virulence.

Abstract: Certain environmental microorganisms can cause severe human infections, even in the absence of an obvious requirement for transition through an animal host for replication (“accidental virulence”). To understand this process, we compared eleven isolate genomes of Burkholderia pseudomallei (Bp), a tropical soil microbe and causative agent of the human and animal disease melioidosis. We found evidence for the existence of several new genes in the Bp reference genome, identifying 282 novel genes supported by at least two independent lines of supporting evidence (mRNA transcripts, database homologs, and presence of ribosomal binding sites) and 81 novel genes supported by all three lines. Within the Bp core genome, 211 genes exhibited significant levels of positive selection (4.5%), distributed across many cellular pathways including carbohydrate and secondary metabolism. Functional experiments revealed that certain positively selected genes might enhance mammalian virulence by interacting with host cellular pathways or utilizing host nutrients. Evolutionary modifications improving Bp environmental fitness may thus have indirectly facilitated the ability of Bp to colonize and survive in mammalian hosts. These findings improve our understanding of the pathogenesis of melioidosis, and establish Bp as a model system for studying the genetics of accidental virulence.

Insect Infection Model for Campylobacter jejuni Reveals That O-methyl Phosphoramidate Has Insecticidal Activity

Abstract: Galleria mellonella (wax moth) larvae have elsewhere been shown to be susceptible to pathogens such as Francisella tularensis, Burkholderia mallei, and Pseudomonas aeruginosa. We report that the larvae are rapidly killed by Campylobacter jejuni at 37 degrees C. Three strains of C. jejuni tested, 11168H (human diarrheal isolate), G1 (human Guillain-Barre syndrome isolate), and 81-176 (human diarrheal isolate), were equally effective at killing G. mellonella larvae. A panel of defined mutants of C. jejuni 11168H, in known or putative virulence genes, showed different degrees of attenuation in G. mellonella larvae. A mutant lacking the O-methyl phosphoramidate (MeOPN) capsule side group was attenuated, clearly demonstrating that MeOPN has a role in virulence. This new model of C. jejuni infection should facilitate the identification of novel virulence genes.

Human immune responses to Burkholderia pseudomallei characterized by protein microarray analysis.

Abstract: The gram-negative bacillus Burkholderia pseudomallei causes human melioidosis, which ranges from a chronic localized infection to severe disseminated infection and often septicemia [1]. This disease occurs in tropical areas, particularly Southeast Asia and northern Australia, but is being increasingly reported in other tropical regions [2, 3]. B. pseudomallei can also establish persistent and asymptomatic infections that can emerge up to 62 years later as fulminant disease [4]. Individuals who recover from the disease may experience subsequent episodes of clinical illness, with 6%–13% of these cases due to reinfection but the majority a consequence of the failure to eliminate the pathogen after treatment with antimicrobials [5]. The host immune responses required to recover from melioidosis or to prevent infection in humans living in melioidosis-endemic areas are largely unknown. With use of a murine model of melioidosis, both cell-mediated and humoral immune responses have been shown to play roles in protection [6]. Cell-mediated responses involving natural killer (NK) cells and adaptive T cells producing interferon-γ (IFN-γ) play an important role in control of infection [7–9]. Our previous studies have revealed that memory CD4+, CD8+T (TEMRA), and NK cells from seropositive healthy individuals living in endemic areas or from individuals who have recovered from melioidosis are primed and produce IFN-γ in vitro in response to killed B. pseudomallei or the bacterial ABC transporter proteins, LolC, OppA, or PotF. The magnitude of these cellular responses correlated with antibody titers to killed B. pseudomallei cells detected by means of conventional indirect hemagglutination assay (IHA) [10]. However, the identity of other antigens recognized by the plasma of these individuals is not known. High-throughput protein microarrays have previously been developed and used to map the humoral responses to individual bacterial and viral proteins [11–16]. Recently, we have devised a B. pseudomallei protein array and probed it with serum specimens from acute melioidosis patients in Northeast Thailand and Singapore. Mapping the profile of antibody responses has allowed the identification of proteins that can be used as serodiagnostic antigens for melioidosis [17]. The potential for these antigens to stimulate cell-mediated immune responses and the identification of proteins that could induce protective immune responses has not been reported. This study aimed to identify B. pseudomallei proteins that could be candidate protective antigens. A B. pseudomallei protein array was probed with plasma from individuals who had recovered from melioidosis after receiving antibiotic therapy and from seropositive individuals living in endemic areas but with no history of melioidosis. We also sought to determine whether recurrent disease, septic disease, or localized infection influenced the antibody response profile and how these antibody responses were related to T cell responses in individuals. In the longer term, our results will support research to devise vaccines against melioidosis.

The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis.

Abstract: Background All four Francisella tularensis subspecies possess gene clusters with potential to express type IV pili (Tfp). These clusters include putative pilin genes, as well as pilB, pilC and pilQ, required for secretion and assembly of Tfp. A hallmark of Tfp is the ability to retract the pilus upon surface contact, a property mediated by the ATPase PilT. Interestingly, out of the two major human pathogenic subspecies only the highly virulent type A strains have a functional pilT gene.

Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243.

Abstract: Burkholderia pseudomallei is an important human pathogen whose infection biology is still poorly understood. The bacterium is endemic to tropical regions, including South East Asia and Northern Australia, where it causes melioidosis, a serious disease associated with both high mortality and antibiotic resistance. B. pseudomallei is a Gram-negative facultative intracellular pathogen that is able to replicate in macrophages. However despite the critical nature of its interaction with macrophages, few anti-macrophage factors have been characterized to date. Here we perform a genome-wide gain of function screen of B. pseudomallei strain K96243 to identify loci encoding factors with anti-macrophage activity. We identify a total of 113 such loci scattered across both chromosomes, with positive gene clusters encoding transporters and secretion systems, enzymes/toxins, secondary metabolite, biofilm, adhesion and signal response related factors. Further phenotypic analysis of four of these regions shows that the encoded factors cause striking cellular phenotypes relevant to infection biology, including apoptosis, formation of actin ‘tails’ and multi-nucleation within treated macrophages. The detailed analysis of the remaining host of loci will facilitate genetic dissection of the interaction of this important pathogen with host macrophages and thus further elucidate this critical part of its infection cycle.

Deletion of the Bacillus anthracis capB homologue in Francisella tularensis subspecies tularensis generates an attenuated strain that protects mice against virulent tularaemia.

Abstract: As there is currently no licensed vaccine against Francisella tularensis, the causative agent of tularaemia, the bacterium is an agent of concern as a potential bioweapon. Although F. tularensis has a low infectious dose and high associated mortality, it possesses few classical virulence factors. An analysis of the F. tularensis subspecies tularensis genome sequence has revealed the presence of a region containing genes with low sequence homology to part of the capBCADE operon of Bacillus anthracis. We have generated an isogenic capB mutant of F. tularensis subspecies tularensis SchuS4 and shown it to be attenuated. Furthermore, using BALB/c mice, we have demonstrated that this capB strain affords protection against significant homologous challenge with the wild-type strain. These data have important implications for the development of a defined and efficacious tularaemia vaccine.

Comparison of a nontoxic variant of Clostridium perfringens α-toxin with the toxic wild-type strain

Abstract: The α-toxin produced by Clostridium perfringens is one of the best-studied examples of a toxic phospholipase C. In this study, a nontoxic mutant protein from C. perfringens strain NCTC8237 in which Thr74 is substituted by isoleucine (T74I) has been characterized and is compared with the toxic wild-type protein. Thr74 is part of an exposed loop at the proposed membrane-interfacing surface of the toxin. The mutant protein had markedly reduced cytotoxic and myotoxic activities. However, this substitution did not significantly affect the catalytic activity towards water-soluble substrate or the overall three-dimensional structure of the protein. The data support the proposed role of the 70-90 loop in the recognition of membrane phospholipids. These findings also provide key evidence in support of the hypothesis that the hydrolysis of both phosphatidylcholine and sphingomyelin are required for the cytolytic and toxic activity of phospholipases.

Insecticide derived from campylobacter bacteria

Abstract: There is provided an insecticide compound comprising a saccharide substituted by at least one O-alkyl phosphoramidate group, particularly a polysaccharide substituted by at least on O-methyl phosphoramidate group. There are also provided methods of assessing the virulence of a Campylobacter bacterium, comprising administering a sample containing the bacterium to an insect and observing whether the insect dies or has impaired activity within a given period.