Reciprocal Analysis of Francisella novicida Infections of a Drosophila melanogaster Model Reveal Host-Pathogen Conflicts Mediated by Reactive Oxygen and imd-Regulated Innate Immune Response

The survival of a bacterial pathogen within a host depends upon its ability to outmaneuver the host immune response. Thus, mutant pathogens provide a useful tool for dissecting host-pathogen relationships, as the strategies the microbe has evolved to counteract immunity reveal a host's immune mechanisms. In this study, we examined the pathogen Francisella novicida and identified new bacterial virulence factors that interact with different parts of the Drosophila melanogaster innate immune system. We performed a genome-wide screen to identify F. novicida genes required for growth and survival within the fly and identified a set of 149 negatively selected mutants. Among these, we identified a class of genes including the transcription factor oxyR, and the DNA repair proteins uvrB, recB, and ruvC that help F. novicida resist oxidative stress. We determined that these bacterial genes are virulence factors that allow F. novicida to counteract the fly melanization immune response. We then performed a second in vivo screen to identify an additional subset of bacterial genes that interact specifically with the imd signaling pathway. Most of these mutants have decreased resistance to the antimicrobial peptide polymyxin B. Characterization of a mutation in the putative transglutaminase FTN_0869 produced a curious result that could not easily be explained using known Drosophila immune responses. By using an unbiased genetic screen, these studies provide a new view of the Drosophila immune response from the perspective of a pathogen. We show that two branches of the fly's immunity are important for fighting F. novicida infections in a model host: melanization and an imd-regulated immune response, and identify bacterial genes that specifically counteract these host responses. Our work suggests that there may be more to learn about the fly immune system, as not all of the phenotypes we observe can be readily explained by its interactions with known immune responses

An O-Antigen glycoconjugate vaccine produced using protein glycan coupling technology is protective in an inhalational rat model of tularemia

There is a requirement for an efficacious vaccine to protect people against infection from Francisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) of F. tularensis is suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugate F. tularensis O-antigen glycans to the immunogenic carrier protein Pseudomonas aeruginosa exoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain of F. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge with F. tularensis Schu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines

Efficacy of cathelicidin-mimetic antimicrobial peptoids against staphylococcus aureus

Staphylococcus aureus is one of the most common pathogens associated with infection in wounds. The current standard of care uses a combination of disinfection and drainage followed by conventional antibiotics such as methicillin. Methicillin and vancomycin resistance has rendered these treatments ineffective, often causing the reemergence of infection. This study examines the use of antimicrobial peptoids (sequence-specific poly-N-substituted glycines) designed to mimic naturally occurring cationic, amphipathic host defense peptides, as an alternative to conventional antibiotics. These peptoids also show efficient and fast (<30 min) killing of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) at low micromolar concentrations without having apparent cytotoxic side effects in vivo. Additionally, these novel peptoids show excellent efficacy against biofilm formation and detachment for both MSSA and MRSA. In comparison, conventional antibiotics were unable to detach or prevent formation of biofilms. One cationic 12mer, Peptoid 1, shows great promise, as it could prevent formation of and detach biofilms at concentrations as low as 1.6 μM. The use of a bioluminescent S. aureus murine incision wound model demonstrated clearance of infection in peptoid-treated mice within 8 days, conveying another advantage these peptoids have over conventional antibiotics. These results provide clear evidence of the potential for antimicrobial peptoids for the treatment of S. aureus wound infections. IMPORTANCE Staphylococcus aureus resistance is a consistent problem with a large impact on the health care system. Infections with resistant S. aureus can cause serious adverse effects and can result in death. These antimicrobial peptoids show efficient killing of bacteria both as a biofilm and as free bacteria, often doing so in less than 30 min. As such, these antimicrobials have the potential to alleviate the burden that Staphylococcus infections have on the health care system and cause better outcomes for infected patients

Drosophila eiger Mutants Are Sensitive to Extracellular Pathogens

We showed previously that eiger, the Drosophila tumor necrosis factor homolog, contributes to the pathology induced by infection with Salmonella typhimurium. We were curious whether eiger is always detrimental in the context of infection or if it plays a role in fighting some types of microbes. We challenged wild-type and eiger mutant flies with a collection of facultative intracellular and extracellular pathogens, including a fungus and Gram-positive and Gram-negative bacteria. The response of eiger mutants divided these microbes into two groups: eiger mutants are immunocompromised with respect to extracellular pathogens but show no change or reduced sensitivity to facultative intracellular pathogens. Hence, eiger helps fight infections but also can cause pathology. We propose that eiger activates the cellular immune response of the fly to aid clearance of extracellular pathogens. Intracellular pathogens, which can already defeat professional phagocytes, are unaffected by eiger

Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survival in vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuation in vivo were identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarked bpsl2248, tex, rpiR, bpsl1728, and bpss1528 deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was tested in vitro and in vivo to confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important to in vivo virulence with roles in different stages of B. pseudomallei pathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and the tex mutant was capable of providing protective immunity against challenge with wild-type B. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates

Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis.

Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l(-1) of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines

Polyelectrolyte Multilayer Nanocoating Dramatically Reduces Bacterial Adhesion to Polyester Fabric

Bacterial adhesion to textiles is thought to contribute to odor and infection. Alternately exposing polyester fabric to aqueous solutions of poly­(diallyldimethylammonium chloride) (PDDA) and poly­(acrylic acid) (PAA) is shown here to create a nanocoating that dramatically reduces bacterial adhesion. Ten PDDA/PAA bilayers (BL) are 180 nm thick and only increase the weight of the polyester by 2.5%. The increased surface roughness and high degree of PAA ionization leads to a surface with a negative charge that causes a reduction in adhesion of <i>Staphylococcus aureus</i> by 50% when compared to uncoated fabric, after rinsing with sterilized water, because of electrostatic repulsion. <i>S. aureus</i> bacterial adhesion was quantified using bioluminescent radiance measured before and after rinsing, revealing 99% of applied bacteria were removed with a ten bilayer PDDA/PAA nanocoating. The ease of processing, and benign nature of the polymers used, should make this technology useful for rendering textiles antifouling on an industrial scale