Low Oxygen Concentration Reduces Neisseria gonorrhoeae Susceptibility to Resazurin.

Neisseria gonorrhoeae has developed resistance to every antibiotic currently approved for the treatment of gonorrhea, prompting the development of new therapies. The phenoxazine dye resazurin exhibits robust antimicrobial activity against N. gonorrhoeae in vitro but fails to limit vaginal colonization by N. gonorrhoeae in a mouse model. The lack of in vivo efficacy may be due to oxygen limitation as in vitro susceptibility assays with resazurin are conducted under atmospheric oxygen while a microaerophilic environment is present in the vagina. Here, we utilized broth microdilution assays to determine the susceptibility of N. gonorrhoeae to resazurin under low and atmospheric oxygen conditions. The minimal inhibitory concentration of resazurin for multiple N. gonorrhoeae clinical isolates was significantly higher under low oxygen. This effect was specific to resazurin as N. gonorrhoeae was equally susceptible to other antibiotics under low and atmospheric oxygen conditions. The reduced susceptibility of N. gonorrhoeae to resazurin under low oxygen was largely attributed to reduced oxidative stress, as the addition of antioxidants under atmospheric oxygen mimicked the reduced susceptibility to resazurin observed under low oxygen. Together, these data suggest oxygen concentration is an important factor to consider when evaluating the efficacy of new antibiotics against N. gonorrhoeae in vitro.

Role of dipA and pilD in Francisella tularensis Susceptibility to Resazurin.

The phenoxazine dye resazurin exhibits bactericidal activity against the Gram-negative pathogens Francisella tularensis and Neisseria gonorrhoeae. One resazurin derivative, resorufin pentyl ether, significantly reduces vaginal colonization by Neisseria gonorrhoeae in a mouse model of infection. The narrow spectrum of bacteria susceptible to resazurin and its derivatives suggests these compounds have a novel mode of action. To identify potential targets of resazurin and mechanisms of resistance, we isolated mutants of F. tularensis subsp. holarctica live vaccine strain (LVS) exhibiting reduced susceptibility to resazurin and performed whole genome sequencing. The genes pilD (FTL_0959) and dipA (FTL_1306) were mutated in half of the 46 resazurin-resistant (RZR) strains sequenced. Complementation of select RZR LVS isolates with wild-type dipA or pilD partially restored sensitivity to resazurin. To further characterize the role of dipA and pilD in resazurin susceptibility, a dipA deletion mutant, ΔdipA, and pilD disruption mutant, FTL_0959d, were generated. Both mutants were less sensitive to killing by resazurin compared to wild-type LVS with phenotypes similar to the spontaneous resazurin-resistant mutants. This study identified a novel role for two genes dipA and pilD in F. tularensis susceptibility to resazurin.

Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells.

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 Role and Mechanism of Erythrocyte Invasion by Francisella tularensis.

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.

Antibacterial activity of resazurin-based compounds against Neisseria gonorrhoeae in vitro and in vivo.

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 exhibits 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.

Temperature-Dependent Gentamicin Resistance of Francisella tularensis is Mediated by Uptake Modulation.

Gentamicin (Gm) is an aminoglycoside commonly used to treat bacterial infections such as tularemia - the disease caused by Francisella tularensis. In addition to being pathogenic, F. tularensis is found in environmental niches such as soil where this bacterium likely encounters Gm producers (Micromonospora sp.). Here we show that F. tularensis exhibits increased resistance to Gm at ambient temperature (26°C) compared to mammalian body temperature (37°C). To evaluate whether F. tularensis was less permeable to Gm at 26°C, a fluorescent marker [Texas Red (Tr)] was conjugated with Gm, yielding Tr-Gm. Bacteria incubated at 26°C showed reduced fluorescence compared to those at 37°C when exposed to Tr-Gm suggesting that uptake of Gm was reduced at 26°C. Unconjugated Gm competitively inhibited uptake of Tr-Gm, demonstrating that this fluorescent compound was taken up similarly to unconjugated Gm. Lysates of F. tularensis bacteria incubated with Gm at 37°C inhibited the growth of Escherichia coli significantly more than lysates from bacteria incubated at 26°C, further indicating reduced uptake at this lower temperature. Other facultative pathogens (Listeria monocytogenes and Klebsiella pneumoniae) exhibited increased resistance to Gm at 26°C suggesting that the results generated using F. tularensis may be generalizable to diverse bacteria. Regulation of the uptake of antibiotics provides a mechanism by which facultative pathogens survive alongside antibiotic-producing microbes in nature.

Genetic engineering of Francisella tularensis LVS for use as a novel live vaccine platform against Pseudomonas aeruginosa infections.

Francisella tularensis LVS (Live Vaccine Strain) is an attenuated bacterium that has been used as a live vaccine. Patients immunized with this organism show a very long-term memory response (over 30 years post vaccination) evidenced by the presence of indicators of robust cell-mediated immunity. Because F. tularensis LVS is such a potent vaccine, we hypothesized that this organism would be an effective vaccine platform. First, we sought to determine if we could genetically modify this strain to produce protective antigens of a heterologous pathogen. Currently, there is not a licensed vaccine against the important opportunistic bacterial pathogen, Pseudomonas aeruginosa. Because many P. aeruginosa strains are also drug resistant, the need for effective vaccines is magnified. Here, F. tularensis LVS was genetically modified to express surface proteins PilAPa, OprFPa, and FliCPa of P. aeruginosa. Immunization of mice with LVS expressing the recombinant FliCPa led to a significant production of antibodies specific for P. aeruginosa. However, mice that had been immunized with LVS expressing PilAPa or OprFPa did not produce high levels of antibodies specific for P. aerugionsa. Therefore, the recombinant LVS strain engineered to produce FliCPa may be able to provide immune protection against a P. aeruginosa challenge. However for future use of this vaccine platform, selection of the appropriate recombinant antigen is critical as not all recombinant antigens expressed in this strain were immunogenic.

The contribution of the glycine cleavage system to the pathogenesis of Francisella tularensis.

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.

The use of resazurin as a novel antimicrobial agent against Francisella tularensis.

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.

Role of NK cells in host defense against pulmonary type A Francisella tularensis infection.

Pneumonic tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Understanding the pulmonary immune response to this bacterium is necessary for developing effective vaccines and therapeutics. In this study, characterization of immune cell populations in the lungs of mice infected with the type A strain Schu S4 revealed a significant loss in natural killer (NK) cells over time. Since this decline in NK cells correlated with morbidity and mortality, we hypothesized these cells contribute to host defense against Schu S4 infection. Depletion of NK cells prior to Schu S4 challenge significantly reduced IFN-γ and granzyme B in the lung but had no effect on bacterial burden or disease progression. Conversely, increasing NK cell numbers with the anti-apoptotic cytokine IL-15 and soluble receptor IL-15Rα had no significant impact on Schu S4 growth in vivo. A modest decrease in median time to death, however, was observed in live vaccine strain (LVS)-vaccinated mice depleted of NK1.1+ cells and challenged with Schu S4. Therefore, NK cells do not appear to contribute to host defense against acute respiratory infection with type A F. tularensis in vivo, but they play a minor role in protection elicited by LVS vaccination.

A Francisella tularensis live vaccine strain that improves stimulation of antigen-presenting cells does not enhance vaccine efficacy.

Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS), does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system.

A Francisella tularensis locus required for spermine responsiveness is necessary for virulence.

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.