Neisseria gonorrhoeae produces no known siderophores but can employ host-derived, iron-binding proteins, including transferrin and lactoferrin, as iron sources. Given the propensity of this pathogen to hijack rather than synthesize iron-sequestering molecules, we hypothesized that the ability to use siderophores produced by other bacteria, or xenosiderophores, may also play a role in the survival of the gonococcus. Among a panel of diverse siderophores, only the catecholate xenosiderophores enterobactin and salmochelin promoted growth of gonococcal strain FA19. Surprisingly, the internalization pathway was independent of TonB or any of the TonB-dependent transporters. Xenosiderophore-mediated growth was similarly independent of the pilinextruding secretin formed by PilQ and of the hydrophobic-agent efflux system composed of MtrCDE. The fbpABC operon encodes a periplasmic-binding-protein-dependent ABC transport system that enables the gonococcus to transport iron into the cell subsequent to outer membrane translocation. We hypothesized that the FbpABC proteins, required for ferric iron transport from transferrin and lactoferrin, might also contribute to the utilization of xenosiderophores as iron sources. We created mutants that conditionally expressed FbpABC from an IPTG-inducible promoter. We determined that expression of FbpABC was required for growth of gonococcal strain FA19 in the presence of enterobactin and salmochelin. The monomeric component of enterobactin, dihydroxybenzoylserine (DHBS), and the S2 form of salmochelin specifically promoted FbpABC-dependent growth of FA19. This study demonstrated that the gonococcal FbpABC transport system is required for utilization of some xenosiderophores as iron sources and that growth promotion by these ferric siderophores can occur in the absence of TonB or individual TonB-dependent transporters.

Survival of Neisseria gonorrhoeae within host epithelial cells is expected to be important in the pathogenesis of gonococcal disease. We previously demonstrated that strain FA1090 derives iron from a host cell in a process that requires the Ton complex and a putative TonB-dependent transporter, TdfF. FA1090, however, lacks the gonococcal genetic island (GGI) that is present in the majority of strains. The GGI in strain MS11 has been partially characterized, and it encodes a type IV secretion system (T4SS) involved in DNA release. In this study we investigated the role of iron acquisition and GGI-encoded gene products in gonococcal survival within cervical epithelial cells. We demonstrated that intracellular survival of MS11 was dependent on acquisition of iron from the host cell, but unlike the findings for FA1090, expression of the Ton complex was not required. Survival was not dependent on a putative TonB-like protein encoded in the GGI but instead was directly linked to T4SS structural components in a manner independent of the ability to release or internalize DNA. These data suggest that expression of selected GGI-encoded open reading frames confers an advantage during cervical cell infection. This study provides the first link between expression of the T4SS apparatus and intracellular survival of gonococci.Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea. Although symptomatic urethritis is the most common presentation in men, asymptomatic infections associated with cervical acquisition in women can lead to ascension of the bacteria to the upper reproductive tract. In this context, an understanding of the immune evasion tactics employed by this organism is essential for eliminating potential downstream sequelae, including disseminated gonococcal infection, pelvic inflammatory disease, infertility, and ectopic pregnancy (23,45). The outcome of gonococcal infections and the resulting clinical manifestations elicited are determined, in part, by strain-specific factors. The differences are expected to impact host-pathogen interactions that occur at the primary site of infection, the urogenital epithelium.Productive gonococcal infections require that the bacteria circumvent the innate iron-withholding mechanisms employed by the human host. Moreover, epithelial cell responses to Neisseria infection, including reduced expression of the transferrin receptor gene and diminished transferrin receptor cycling, which result in further limitation of iron availability, have been described previously (8,9,34). As the ability to acquire iron from the human host is a key determinant in gonococcal pathogenesis (1,15), N. gonorrhoeae has evolved numerous mechanisms to acquire this required nutrient in an iron-depleted environment. Various iron uptake systems that are thought to promote gonococcal replication in extracellular niches have been characterized (42). Specifically, these systems hijack iron from the host proteins transferrin (14), lactoferrin (7), and hemoglobin (12) in a ma...

Pregnant mares serum gonadotrophin (PMSG) was used in combination with prostaglandin F2alpha or its analogues to induce superovulation in 25 heifers. Total unconjugated oestrogen and progesterone were determined in peripheral plasma of these superovulated animals, and the levels compared with those found during the normal oestrous cycle. A very high level of oestrogen was found between day 3 and 6 after superovulation, and it seems likely that large unovulated follicles were responsible for the excess steroid. Similarly, progesterone levels were raised in the superovulated animal presumably due to production by the numerous corpora lutea. In two heifers, in which overstimulation of follicular development had occurred, there were no ovulations in one of the animals while in the other animal there were 16 ovulations but early regression of corpora lutea was indicated. It is concluded that the poor recovery of eggs on day 6 after superovulation could have been due to deleterious effects of high levels of oestrogen on either the motility of the genital tract causing the premature transport of eggs, or the properties of the zona pellucida.

ObjectiveImmune suppression during critical illness predisposes to serious infections. We sought to determine the mechanisms regulating tolerance and cross-tolerance to common pro-inflammatory danger signals in a model that recapitulates the intact in vivo immune response.Materials and MethodsFlt3-expanded splenocytes obtained from wild-type or matching IRAK-M knockout (IRAK-M-/-), C57BL/6, male mice (8–10 weeks old) were treated repeatedly or alternately with either LPS or CpGA DNA, agonists of Toll-like receptor (TLR)-4 and -9, respectively, over successive 24-hour periods. Supernatants were collected following each 24-hour period with cytokine release (ELISA) and splenocyte IRAK-M expression (Western blot) determined. Tolerance and cross-tolerance were assessed in the absence or presence of programmed death receptor (PD)-1 blocking antibody or IL-7 pre-treatment.Main ResultsSplenocytes notably exhibited both tolerance and cross-tolerance to subsequent treatments with either LPS or CpGA DNA. The character of tolerance and cross-tolerance in this model was distinct following initial LPS or CpGA treatment in that TNFα and IFNγ release (not IL-10) were suppressed following LPS; whereas, initial CpGA treatment suppressed TNFα, IFNγ and IL-10 release in response to subsequent stimulation (LPS or CpGA). Tolerance and cross-tolerance were unrelated to IL-10 release or PD-1 but were attenuated in IRAK-M-/- splenocytes. IL-7 significantly suppressed IRAK-M expression and restored TNFα and IFNγ production without influencing IL-10 release.ConclusionsIn summary, acute immune tolerance and cross-tolerance in response to LPS or CpGA were distinct in that LPS selectively suppressed pro-inflammatory cytokine responses; whereas, CpGA suppressed both pro- and anti-inflammatory responses. The induction of tolerance and cross-tolerance in response to common danger signals was mechanistically unrelated to IL-10 or PD-1 but was directly influenced by IRAK-M expression. IL-7 reduced IRAK-M expression and attenuated immune tolerance induced by either LPS or CpGA, and thus may be useful for reversal of immune tolerance in the setting of critical illness.

OBJECTIVE To evaluate the effects of damage-associated molecular patterns (DAMPs) derived from disrupted mitochondria on canine splenocytes and other immune cells. SAMPLES Liver, spleen, and bone marrow samples obtained from 8 cadavers of healthy research Beagles that had been euthanized for other purposes. PROCEDURES Mitochondria were obtained from canine hepatocytes, and mitochondrial DAMPs (containing approx 75% mitochondrial proteins) were prepared. Mitochondrial DAMPs and the nuclear cytokine high-mobility group box protein 1 were applied to splenocytes, bone marrow-differentiated dendritic cells, and a canine myelomonocytic cell (DH82) line for 6 or 24 hours. Cell culture supernatants from splenocytes, dendritic cells, and DH82 cells were assayed for tumor necrosis factor α with an ELISA. Expression of tumor necrosis factor α mRNA in splenocytes was evaluated with a quantitative real-time PCR assay. RESULTS In all cell populations evaluated, production of tumor necrosis factor α was consistently increased by mitochondrial DAMPs at 6 hours (as measured by an ELISA). In contrast, high-mobility group box protein 1 did not have any independent proinflammatory effects in this experimental system. CONCLUSIONS AND CLINICAL RELEVANCE The study revealed an in vitro inflammatory effect of mitochondrial DAMPs (containing approx 75% mitochondrial proteins) in canine cells and validated the use of an in vitro splenocyte model to assess DAMP-induced inflammation in dogs. This experimental system may aid in understanding the contribution of DAMPs to sepsis and the systemic inflammatory response syndrome in humans. Further studies in dogs are needed to validate the biological importance of these findings and to evaluate the in vivo role of mitochondrial DAMPs in triggering and perpetuating systemic inflammatory states.