Immunization with the C-domain of α-toxin prevents lethal infection, localizes tissue injury, and promotes host response to challenge with Clostridium perfringens

© 2004 by the Infectious Diseases Society of America. All rights reserved.Clostridium perfringens gas gangrene is characterized by rapid tissue destruction, impaired host response, and, often, death. Phospholipase C (α-toxin) is the virulence factor most responsible for these pathologies. The present study investigated the efficacy of active immunization with the C-terminal domain of α-toxin (Cpa247–370) in a murine model of gas gangrene. Primary end points of the study were survival, progression of infection, and tissue perfusion. Secondary end points, which were based on findings of histologic evaluation of tissues, included the extent of tissue destruction and microvascular thrombosis, as well as the magnitude of the tissue inflammatory response. Survival among C-domain–immunized animals was significantly greater than that among sham-immunized control animals. Furthermore, immunization with the C-domain localized the infection and prevented ischemia of the feet. Histopathologic findings demonstrated limited muscle necrosis, reduced microvascular thrombosis, and enhanced granulocytic influx in C-domain–immunized mice. We conclude that immunization with the C-domain of phospholipase C is a viable strategy for the prevention of morbidity and mortality associated with C. perfringens gas gangrene

Genome Sequence of Staphylococcus aureus Ex1, Isolated from a Patient with Spinal Osteomyelitis.

Here, we present the genome sequence of Staphylococcus aureus Ex1, isolated in 2015 from a patient with spinal osteomyelitis at the Royal Devon and Exeter Hospital in the United Kingdom. The availability of the Ex1 genome sequence provides a resource for studying the basis for spinal infection and horizontal gene transfer in S. aureus.This article is freely available via Open Access. Click on the Additional Link above to access the full-text via the publisher's site

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

Diversity of Francisella Species in Environmental Samples from Martha’s Vineyard, Massachusetts

We determined whether Francisella spp. are present in water, sediment, and soil from an active tularemia natural focus on Martha’s Vineyard, Massachusetts, during a multiyear outbreak of pneumonic tularemia. Environmental samples were tested by polymerase chain reaction (PCR) targeting Francisella species 16S rRNA gene and succinate dehydrogenase A (sdhA) sequences; evidence of the agent of tularemia was sought by amplification of Francisella tularensis-specific sequences for the insertion element ISFTu2, 17-kDa protein gene tul4, and the 43-kDa outer membrane protein gene fopA. Evidence of F. tularensis subsp. tularensis, the causative agent of the human infections in this outbreak, was not detected from environmental samples despite its active transmission among ticks and animals in the sampling site. Francisella philomiragia was frequently detected from a brackish-water pond using Francisella species PCR targets, and subsequently F. philomiragia was isolated from an individual brackish-water sample. Distinct Francisella sp. sequences that are closely related to F. tularensis and Francisella novicida were detected from samples collected from the brackish-water pond. We conclude that diverse Francisella spp. are present in the environment where human cases of pneumonic tularemia occur

Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia

Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia

Clusters versus Affinity-Based Approaches in F. tularensis Whole Genome Search of CTL Epitopes

Deciphering the cellular immunome of a bacterial pathogen is challenging due to the enormous number of putative peptidic determinants. State-of-the-art prediction methods developed in recent years enable to significantly reduce the number of peptides to be screened, yet the number of remaining candidates for experimental evaluation is still in the range of ten-thousands, even for a limited coverage of MHC alleles. We have recently established a resource-efficient approach for down selection of candidates and enrichment of true positives, based on selection of predicted MHC binders located in high density “hotspots" of putative epitopes. This cluster-based approach was applied to an unbiased, whole genome search of Francisella tularensis CTL epitopes and was shown to yield a 17–25 fold higher level of responders as compared to randomly selected predicted epitopes tested in Kb/Db C57BL/6 mice. In the present study, we further evaluate the cluster-based approach (down to a lower density range) and compare this approach to the classical affinity-based approach by testing putative CTL epitopes with predicted IC50 values of <10 nM. We demonstrate that while the percent of responders achieved by both approaches is similar, the profile of responders is different, and the predicted binding affinity of most responders in the cluster-based approach is relatively low (geometric mean of 170 nM), rendering the two approaches complimentary. The cluster-based approach is further validated in BALB/c F. tularensis immunized mice belonging to another allelic restriction (Kd/Dd) group. To date, the cluster-based approach yielded over 200 novel F. tularensis peptides eliciting a cellular response, all were verified as MHC class I binders, thereby substantially increasing the F. tularensis dataset of known CTL epitopes. The generality and power of the high density cluster-based approach suggest that it can be a valuable tool for identification of novel CTLs in proteomes of other bacterial pathogens

Whole-Genome Immunoinformatic Analysis of F. tularensis: Predicted CTL Epitopes Clustered in Hotspots Are Prone to Elicit a T-Cell Response

The cellular arm of the immune response plays a central role in the defense against intracellular pathogens, such as F. tularensis. To date, whole genome immunoinformatic analyses were limited either to relatively small genomes (e.g. viral) or to preselected subsets of proteins in complex pathogens. Here we present, for the first time, an unbiased bacterial global immunoinformatic screen of the 1740 proteins of F. tularensis subs. holarctica (LVS), aiming at identification of immunogenic peptides eliciting a CTL response. The very large number of predicted MHC class I binders (about 100,000, IC50 of 1000 nM or less) required the design of a strategy for further down selection of CTL candidates. The approach developed focused on mapping clusters rich in overlapping predicted epitopes, and ranking these “hotspot” regions according to the density of putative binding epitopes. Limited by the experimental load, we selected to screen a library of 1240 putative MHC binders derived from 104 top-ranking highly dense clusters. Peptides were tested for their ability to stimulate IFNγ secretion from splenocytes isolated from LVS vaccinated C57BL/6 mice. The majority of the clusters contained one or more CTL responder peptides and altogether 127 novel epitopes were identified, of which 82 are non-redundant. Accordingly, the level of success in identification of positive CTL responders was 17–25 fold higher than that found for a randomly selected library of 500 predicted MHC binders (IC50 of 500 nM or less). Most proteins (ca. 2/3) harboring the highly dense hotspots are membrane-associated. The approach for enrichment of true positive CTL epitopes described in this study, which allowed for over 50% increase in the dataset of known T-cell epitopes of F. tularensis, could be applied in immunoinformatic analyses of many other complex pathogen genomes

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 3.9Å. 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 oligomerisation 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

Host Factors Required for Modulation of Phagosome Biogenesis and Proliferation of Francisella tularensis within the Cytosol

Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of ∼21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals