Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

Tularemia is caused by the Gram-negative bacterial pathogen Francisella tularensis. Infection of macrophages and their subsequent death are believed to play important roles in the progression of disease. Because complement is a particularly effective opsonin for Francisella, we asked whether complement-dependent uptake of F. tularensis strain SCHU S4 affects the survival of primary human macrophages during infection. Complement component C3 was found to be an essential opsonin in human serum not only for greatly increased uptake of SCHU S4 but also for the induction of macrophage death. Single-cell analysis also revealed that macrophage death did not require a high intracellular bacterial burden. In the presence of C3, macrophage death was observed at 24 h postinfection in a quarter of the macrophages that contained only 1 to 5 bacterial cells. Macrophages infected in the absence of C3 rarely underwent cell death, even when they contained large numbers of bacteria. The need for C3, but not extensive replication of the pathogen, was confirmed by infections with SCHU S4 ΔpurMCD, a mutant capable of phagosome escape but of only limited cytosolic replication. C3-dependent Francisella uptake alone was insufficient to induce macrophage death, as evidenced by the failure of the phagosome escape-deficient mutant SCHU S4 ΔfevR to induce cell death despite opsonization with C3. Together, these findings indicate that recognition of C3-opsonized F. tularensis, but not extensive cytosolic replication, plays an important role in regulating macrophage viability during intracellular infections with type A F. tularensis. KEYWORDS C3, Francisella tularensis, cell death, complement, macrophage F rancisella tularensis is the causative agent of the zoonotic life-threatening disease tularemia. Among the subspecies responsible for disease in human beings, F. tularensis subsp. tularensis (type A) is the most virulent and causes high morbidity and mortality when delivered via the respiratory route (1-4). Very low minimum infectious doses have been reported with pulmonary challenge in humans (2, 5). In mice, alveolar macrophages are among the earliest cells infected following respiratory challenge (6-8). F. tularensis rapidly disrupts the phagosome and enters the cytosol of cultured macrophages, where it replicates to high intracellular numbers (9-11). Likewise, phagocytic cell death and bacteremia follow infection of mice with F. tularensis and lead to secondary colonization and pathology in the spleen, liver, and draining lymph nodes (12-15). Indeed, one of the hallmark histopathological features of disseminated tularemia caused by the F. tularensis subsp. tularensis is the appearance of infected clusters of macrophages and myeloid cells (microgranulomas), which rapidly transform into necrotic foci (13)(14)(15)(16). Infection of macrophages with the laboratory strain F. tularensis subsp. tularensis SCHU S4 does not appear to activate caspase-1-mediated pyroptosis (17, 18), as has been reported for Francisella novicida strain U...

Francisella tularensis has developed a number of effective evasion strategies to counteract host immune defenses, not the least of which is its ability to interact with the complement system to its own advantage. Following exposure of the bacterium to fresh human serum, complement is activated and C3b and iC3b can be found covalently attached to the bacterial surface. However, the lipopolysaccharide and capsule of the F. tularensis cell wall prevent complement-mediated lysis and endow the bacterium with serum resistance. Opsonization of F. tularensis with C3 greatly increases its uptake by human neutrophils, dendritic cells and macrophages. Uptake occurs by an unusual looping morphology in human macrophages. Complement receptor 3 is thought to play an important role in opsonophagocytosis by human macrophages, and signaling through this receptor can antagonize Toll-like receptor 2-initiated macrophage activation. Complement C3 also determines the survival of infected human macrophages and perhaps other cell types. C3-opsonization of F. tularensis subsp. tularensis strain SCHU S4 results in greatly increased death of infected human macrophages, which requires more than complement receptor engagement and is independent of the intracellular replication by the pathogen. Given its entry into the cytosol of host cells, F. tularensis has the potential for a number of other complement-mediated interactions. Studies on the uptake C3-opsonized adenovirus have suggested the existence of a C3 sensing system that initiates cellular responses to cytosolic C3b present on invading microbes. Here we propose that C3 peptides enter the cytosol of human macrophages following phagosome escape of F. tularensis and are recognized as intruding molecular patterns that signal host cell death. With the discovery of new roles for intracellular C3, a better understanding of tularemia pathogenesis is likely to emerge.

Bacteriocins are antimicrobial peptides produced by bacteria while antibiotics are non‐proteinacous small organic antimicrobial molecules. They also differ from antibiotics in modes of action. Their wide range of molecular and functional characteristics include: molecular weight, structure, temperature/pH stability, and killing spectrums of susceptible species. Bacteriocins present potential for diverse applications as treatment/prevention of infectious diseases, food preservers, ect. In the spring of 2006, a bacteriocin‐like substance, currently referred to as Bacteriocin X (Bac. X), was discovered from a putative strain of Bacillus amyloliquefaciens. The objective of this study was to discern the functional and molecular characteristics of Bac. X. It demonstrated a wide spectrum of activity against an array of bacterial organisms (both Gram(+) and Gram(−)), including a variety of potential pathogens. It displayed stability under extreme temperature and pH conditions. It was not notably susceptible to Proteinase K or Trypsin; but its activity was destroyed by Pronase E. Bac. X was also able to destroy mammalian cells. This study is still pursuing the purification of Bac. X from cell filtrate or perhaps a plasmid isolation of its gene to facilitate sequencing and homology comparisons, looking for the identification of the bacteriocin or documentation of a new one.MWSU Summer Research Institute

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