Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S . marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.

The highly infectious and deadly pathogen, Francisella tularensis , is classified by the CDC as a Category A bioterrorism agent. Inhalation of a single bacterium results in an acute pneumonia with a 30–60% mortality rate without treatment. Due to the prevalence of antibiotic resistance, there is a strong need for new types of antibacterial drugs. Resazurin is commonly used to measure bacterial and eukaryotic cell viability through its reduction to the fluorescent product resorufin. When tested on various bacterial taxa at the recommended concentration of 44 μM, a potent bactericidal effect was observed against various Francisella and Neisseria species, including the human pathogens type A F. tularensis (Schu S4) and N. gonorrhoeae . As low as 4.4 μM resazurin was sufficient for a 10-fold reduction in F. tularensis growth. In broth culture, resazurin was reduced to resorufin by F. tularensis . Resorufin also suppressed the growth of F. tularensis suggesting that this compound is the biologically active form responsible for decreasing the viability of F. tularensis LVS bacteria. Replication of F. tularensis in primary human macrophages and non-phagocytic cells was abolished following treatment with 44 μM resazurin indicating this compound could be an effective therapy for tularemia in vivo .

Our purpose was to determine if an acute bout of heading soccer balls adversely affected postural control and self-reported symptoms of cerebral concussion. Thirty-one college-aged soccer players were randomly placed into either a kicking group or a heading group. Subjects either kicked or headed 18 soccer balls over the course of 40 minutes. Subjects had their postural control assessed while standing on a force plate and completed a concussion symptoms checklist on three separate occasions: prior to, immediately following, and 24 hours after kicking or heading. There were no significant differences between the heading and kicking groups on the postural control measures prior to, immediately following, and 24 hours after kicking/heading. The heading group did, however, report significantly more concussion symptoms than the kicking group immediately after heading, but not 24 hours after heading. The number of previous concussions sustained by subjects did not influence the effects of heading. An acute bout of soccer heading appears to cause an increase in self-reported symptoms of cerebral concussion lasting less than 24 hours but no quantifiable deficits in postural control. Further research is needed to investigate the cumulative effects of soccer heading on postural control and other objective measures of brain function.

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.

The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity. In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified. We partially purified and characterized farnesyl-protein transferase from tomato

Neisseria gonorrhoeae is the cause of the second most common sexually transmitted bacterial infection, with ca. 80 million new cases of gonorrhoea reported annually. The recent emergence of clinical isolates resistant to the last monotherapy against this bacterium, the cephalosporins, illustrates the need for new antigonococcal agents. Here we have characterised a new group of antimicrobials based on the compound resazurin that exhibit robust activity against N. gonorrhoeae in vitro. Resazurin inhibits the growth of a broad range of N. gonorrhoeae isolates, including those resistant to multiple antibiotics. Furthermore, treatment of human endometrial cells infected with N. gonorrhoeae with resazurin significantly reduces the number of intracellular bacteria. Whilst resazurin exhibited potent in vitro antimicrobial activity, in vivo resazurin did not limit the colonisation of mice with N. gonorrhoeae following vaginal infection. The ineffectiveness of resazurin in vivo is likely due to its interaction with serum albumin, which completely diminishes its antimicrobial activity. However, treatment of mice with a resazurin analogue (resorufin pentyl ether) that maintains its antimicrobial activity in the presence of serum albumin approached a significant decrease in the percentage of mice vaginally colonised. This treatment also decreased vaginal colonisation by N. gonorrhoeae over time. Together, these data suggest that resazurin derivatives have potential for the treatment of gonorrhoea.

Biosynthesis and acquisition of nutrients during infection are integral to pathogenesis. Members of a metabolic pathway, the glycine cleavage system, have been identified in virulence screens of the intracellular bacterium Francisella tularensis but their role in pathogenesis remains unknown. This system generates 5,10-methylenetetrahydrofolate, a precursor of amino acid and DNA synthesis, from glycine degradation. To characterize this pathway, deletion of the gcvT homolog, an essential member of this system, was performed in attenuated and virulent F. tularensis strains. Deletion mutants were auxotrophic for serine but behaved similar to wild-type strains with respect to host cell invasion, intracellular replication, and stimulation of TNF-α. Unexpectedly, the glycine cleavage system was required for the pathogenesis of virulent F. tularensis in a murine model. Deletion of the gcvT homolog delayed mortality and lowered bacterial burden, particularly in the liver and bloodstream. To reconcile differences between the cell culture model and animal model, minimal tissue culture media was employed to mimic the nutritionally limiting environment of the host. This reevaluation demonstrated that the glycine cleavage system contributes to the intracellular replication of virulent F. tularensis in serine limiting environments. Thus, the glycine cleavage system is the serine biosynthetic pathway of F. tularensis and contributes to pathogenesis in vivo.