Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity.

The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10-3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.

Interactions Between Pseudomonas Immunotoxins and the Plasma Membrane: Implications for CAT-8015 Immunotoxin Therapy.

Acute Lymphoblastic Leukemia (ALL) remains the most frequent cause of cancer-related mortality in children and novel therapies are needed for the treatment of relapsed/refractory childhood ALL. One approach is the targeting of ALL blasts with the Pseudomonas immunotoxin CAT-8015. Although CAT-8015 has potent anti-leukemia activity, with a 32% objective response rate in a phase 1 study of childhood ALL, haemolytic-uremic syndrome (HUS) and vascular leak syndrome (VLS), major dose-limiting toxicities, have limited the use of this therapeutic approach in children. Investigations into the pathogenesis of CAT-8015-induced HUS/VLS are hindered by the lack of an adequate model system that replicates clinical manifestations, but damage to vascular endothelial cells (ECs) and blood cells are believed to be major initiating factors in both syndromes. Since there is little evidence that murine models replicate human HUS/VLS, and CAT-8015-induced HUS/VLS predominantly affects children, we developed human models and used novel methodologies to investigate CAT-8015 interactions with red blood cells (RBCs) from pediatric ALL patients and ECs of excised human mesenteric arteries. We provide evidence that CAT-8015 directly interacts with RBCs, mediated by Pseudomonas toxin. We also show correlation between the electrical properties of the RBC membrane and RBC susceptibility to CAT-8015-induced lysis, which may have clinical implication. Finally, we provide evidence that CAT-8015 is directly cytototoxic to ECs of excised human mesenteric arteries. In conclusion, the human models we developed constitutes the first, and very important, step in understanding the origins of HUS/VLS in immunotoxin therapy and will allow further investigations of HUS/VLS pathogenesis.

Variant proteins stimulate more IgM+ GC B-cells revealing a mechanism of cross-reactive recognition by antibody memory.

Vaccines induce memory B-cells that provide high affinity secondary antibody responses to identical antigens. Memory B-cells can also re-instigate affinity maturation, but how this happens against antigenic variants is poorly understood despite its potential impact on driving broadly protective immunity against pathogens such as Influenza and Dengue. We immunised mice sequentially with identical or variant Dengue-virus envelope proteins and analysed antibody and germinal-centre (GC) responses. Variant protein boosts induced GCs with a higher proportion of IgM+ B cells. The most variant protein re-stimulated GCs with the highest proportion of IgM+ cells with the most diverse, least mutated V-genes and with a slower but efficient serum antibody response. Recombinant antibodies from GC B-cells showed a higher affinity for the variant antigen than antibodies from a primary response, confirming a memory origin. This reveals a new process of antibody memory, that IgM memory cells with fewer mutations participate in secondary responses to variant antigens, demonstrating how the hierarchical structure of B-cell memory is used and indicating the potential and limits of cross-reactive antibody based immunity.

A proteasome inhibitor produced by Burkholderia pseudomallei modulates intracellular growth.

The NRPS/PKS cluster encodes the enzymes necessary for glidobactin synthesis it is partially conserved in various members of the Burkholderia genus including B. pseudomallei. In this study we have shown that the insertional inactivation or deletion of glbC in this cluster in B. pseudomallei could reduce the ability of the bacterium to survive or grow in murine macrophages or in human neutrophils. Exogenously added proteasome inhibitors were able to chemically complement the mutation. The insertional inactivation or deletion of glbC increased virulence in an acute model of infection in Balb/c or C57BL/6 mice but virulence in a chronic model of infection was similar to that of the wild type. Our findings contrast with the previous finding that inactivation of the glb gene cluster in B. pseudomallei strain 1026b resulted in marked attenuation, and provides evidence of differential roles for some genes in virulence of different strains of B. pseudomallei.

Trehalase plays a role in macrophage colonization and virulence of Burkholderia pseudomallei in insect and mammalian hosts.

Trehalose is a disaccharide formed from two glucose molecules. This sugar molecule can be isolated from a range of organisms including bacteria, fungi, plants and invertebrates. Trehalose has a variety of functions including a role as an energy storage molecule, a structural component of glycolipids and plays a role in the virulence of some microorganisms. There are many metabolic pathways that control the biosynthesis and degradation of trehalose in different organisms. The enzyme trehalase forms part of a pathway that converts trehalose into glucose. In this study we set out to investigate whether trehalase plays a role in both stress adaptation and virulence of Burkholderia pseudomallei. We show that a trehalase deletion mutant (treA) had increased tolerance to thermal stress and produced less biofilm than the wild type B. pseudomallei K96243 strain. We also show that the ΔtreA mutant has reduced ability to survive in macrophages and that it is attenuated in both Galleria mellonella (wax moth larvae) and a mouse infection model. This is the first report that trehalase is important for bacterial virulence.

Mapping epigenetic changes to the host cell genome induced by Burkholderia pseudomallei reveals pathogen-specific and pathogen-generic signatures of infection.

The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection.

From crystal structure to in silico epitope discovery in the Burkholderia pseudomallei flagellar hook-associated protein FlgK.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a potentially fatal infection that is endemic in Southeast Asia and Northern Australia that is poorly controlled by antibiotics. Research efforts to identify antigenic components for a melioidosis vaccine have led to the identification of several proteins, including subunits forming the flagella that mediate bacterial motility, host colonization, and virulence. This study focuses on the B. pseudomallei flagellar hook-associated protein (FlgK(Bp)), and provides the first insights into the 3D structure of FlgK proteins as targets for structure-based antigen engineering. The FlgK(Bp) crystal structure (presented here at 1.8-Å resolution) reveals a multidomain fold, comprising two small ß-domains protruding from a large elongated α-helical bundle core. The evident structural similarity to flagellin, the flagellar filament subunit protein, suggests that, depending on the bacterial species, flagellar hook-associated proteins are likely to show a conserved, elongated α-helical bundle scaffold coupled to a variable number of smaller domains. Furthermore, we present immune serum recognition data confirming, in agreement with previous findings, that recovered melioidosis patients produce elevated levels of antibodies against FlgK(Bp), in comparison with seronegative and seropositive healthy subjects. Moreover, we show that FlgK(Bp) has cytotoxic effects on cultured murine macrophages, suggesting an important role in bacterial pathogenesis. Finally, computational epitope prediction methods applied to the FlgK(Bp) crystal structure, coupled with in vitro mapping, allowed us to predict three antigenic regions that locate to discrete protein domains. Taken together, our results point to FlgK(Bp) as a candidate for the design and production of epitope-containing subunits/domains as potential vaccine components.

Identification of a key residue for oligomerisation and pore-formation of Clostridium perfringens NetB.

Necrotic enteritis toxin B (NetB) is a ß-pore-forming toxin produced by Clostridium perfringens and has been identified as a key virulence factor in the pathogenesis of avian necrotic enteritis, a disease causing significant economic damage to the poultry industry worldwide. In this study, site-directed mutagenesis was used to identify amino acids that play a role in NetB oligomerisation and pore-formation. NetB K41H showed significantly reduced toxicity towards LMH cells and human red blood cells relative to wild type toxin. NetB K41H was unable to oligomerise and form pores in liposomes. These findings suggest that NetB K41H could be developed as a genetic toxoid vaccine to protect against necrotic enteritis.

Lipoprotein CD0873 is a novel adhesin of Clostridium difficile.

Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduction in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed significantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease.

Protection against avian necrotic enteritis after immunisation with NetB genetic or formaldehyde toxoids.

NetB (necrotic enteritis toxin B) is a recently identified ß-pore-forming toxin produced by Clostridium perfringens. This toxin has been shown to play a major role in avian necrotic enteritis. In recent years, a dramatic increase in necrotic enteritis has been observed, especially in countries where the use of antimicrobial growth promoters in animal feedstuffs has been banned. The aim of this work was to determine whether immunisation with a NetB toxoid would provide protection against necrotic enteritis. The immunisation of poultry with a formaldehyde NetB toxoid or with a NetB genetic toxoid (W262A) resulted in the induction of antibody responses against NetB and provided partial protection against disease.

The ability of CpG oligonucleotides to protect mice against Francisella tularensis live vaccine strain but not fully virulent F. tularensis subspecies holarctica is reflected in cell-based assays.

CpG DNA is a potent activator of the innate immune system. Here the protective effects of CpG DNA are assessed against the facultative intracellular pathogen Francisella tularensis. Dosing of mice with CpG DNA provided protection against disease caused by F. tularensis subsp. holarctica live vaccine strain (LVS) but did not protect against the fully virulent F. tularensis subsp holarctica strain HN63. Similarly, in vitro studies in J774A murine macrophage-like cells demonstrated that stimulation with CpG DNA enables control of intracellular replication of LVS but not HN63. These data confirm findings that CpG DNA may have limited efficacy in providing protection against fully virulent strains of F. tularensis and also suggest that in vitro assays may be useful for the evaluation of novel treatments for virulent F. tularensis.

Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens.

NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.

Role of RelA and SpoT in Burkholderia pseudomallei virulence and immunity.

Burkholderia pseudomallei is a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created a relA spoT double mutant in B. pseudomallei strain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotype in vitro and in vivo. The B. pseudomallei ΔrelA ΔspoT mutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in the Galleria mellonella insect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the ΔrelA ΔspoT mutant resulted in partial protection against infection with wild-type B. pseudomallei. In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation in B. pseudomallei, and the ΔrelA ΔspoT mutant may be a promising live-attenuated vaccine candidate.

Low-dose exposure of C57BL/6 mice to burkholderia pseudomallei mimics chronic human melioidosis.

Burkholderia pseudomallei is the etiological agent of human melioidosis, a disease with a broad spectrum of clinical manifestations ranging from fatal septicemia to chronic localized infection or asymptomatic latent infection. Most clinical and immunological studies to date have focused on the acute disease process; however, little is known about pathology and immune response in chronic melioidosis. Here, we have developed a murine model of chronic disease by challenging C57BL/6 mice intranasally with a low dose of B. pseudomallei and monitoring them up to 100 days postinfection. Bacterial burdens were heterogeneous in different animals at all time points, consistent with the spectrum of clinical severity observed in humans. Proinflammatory cytokines such as gamma interferon (IFN-γ), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) were induced during chronic infection, and histopathological analysis showed features in common with human melioidosis. Interestingly, many of these features were similar to those induced by Mycobacterium tuberculosis in humans, such as development of a collagen cord that encapsulates the lesions, the presence of multinucleated giant cells, and granulomas with a caseous necrotic center, which may explain why chronic melioidosis is often misdiagnosed as tuberculosis. Our model now provides a relevant and practical tool to define the immunological features of chronic melioidosis and aid in the development of more effective treatment of this disease in humans.

Superoxide dismutase C is required for intracellular survival and virulence of Burkholderia pseudomallei.

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a life-threatening disease of humans. Within host cells, superoxide is an important mediator of pathogen killing. In this study, we have identified the B. pseudomallei K96243 sodC gene, shown that it has superoxide dismutase activity, and constructed an allelic deletion mutant of this gene. Compared with the wild-type, the mutant was more sensitive to killing by extracellular superoxide, but not to superoxide generated intracellularly. The sodC mutant showed a markedly decreased survival in J774A.1 mouse macrophages, and reduced numbers of bacteria were recovered from human polymorphonuclear neutrophils (PMNs) when compared with the wild-type. The numbers of wild-type or mutant bacteria recovered from human diabetic neutrophils were significantly lower than from normal human neutrophils. The sodC mutant was attenuated in BALB/c mice. Our results indicate that SodC plays a key role in the virulence of B. pseudomallei, but that diabetics are not more susceptible to infection because of a reduced ability of PMNs to kill by superoxide.

Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis.

BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella) infection models. RESULTS: B. pseudomallei strains 576 and K96243, which have low median lethal dose (MLD) values in mice, were able to replicate and induce cellular damage in macrophages and caused rapid death of G. mellonella. In contrast, B. pseudomallei strain 708a, which is attenuated in mice, showed reduced replication in macrophages, negligible cellular damage and was avirulent in G. mellonella larvae. B. thailandensis isolates were less virulent than B. pseudomallei in all of the models tested. However, we did record strain dependent differences. B. oklahomensis isolates were the least virulent isolates. They showed minimal ability to replicate in macrophages, were unable to evoke actin-based motility or to form multinucleated giant cells and were markedly attenuated in G. mellonella compared to B. thailandensis. CONCLUSIONS: We have shown that the alternative infection models tested here, namely macrophages and Galleria mellonella, are able to distinguish between strains of B. pseudomallei, B. thailandensis and B. oklahomensis and that these differences reflect the observed virulence in murine infection models. Our results indicate that B. oklahomensis is the least pathogenic of the species investigated. They also show a correlation between isolates of B. thailandensis associated with human infection and virulence in macrophage and Galleria infection models.

Galleria mellonella as an infection model for Campylobacter jejuni virulence.

Larvae of Galleria mellonella (Greater Wax Moth) have been shown to be susceptible to Campylobacter jejuni infection and our study characterizes this infection model. Following infection with C. jejuni human isolates, bacteria were visible in the haemocoel and gut of challenged larvae, and there was extensive damage to the gut. Bacteria were found in the extracellular and cell-associated fraction in the haemocoel, and it was shown that C. jejuni can survive in insect cells. Finally, we have used the model to screen a further 67 C. jejuni isolates belonging to different MLST types. Isolates belonging to ST257 were the most virulent in the Galleria model, whereas those belonging to ST21 were the least virulent.

Yersinia pseudotuberculosis mntH functions in intracellular manganese accumulation, which is essential for virulence and survival in cells expressing functional Nramp1.

Manganese has an important yet undefined role in the virulence of many bacterial pathogens. In this study we confirm that a null mutation in Yersinia pseudotuberculosis mntH reduces intracellular manganese accumulation. An mntH mutant was susceptible to killing by reactive oxygen species when grown under manganese-limited conditions. The mntH mutant was defective in survival and growth in macrophages expressing functional Nramp1, but in macrophages deficient in Nramp the bacteria were able to survive and replicate. In Galleria mellonella, the mntH mutant was attenuated. Taken together, these data suggest a role for manganese in Y. pseudotuberculosis during macrophage intracellular survival, protecting the bacteria from the antimicrobial products released during the respiratory burst.

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

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.