Ann E. Jerse

A gonococcal efflux pump system enhances bacterial survival in a female mouse model of genital tract infection

ABSTRACT Active efflux of antimicrobial substances is likely to be an important bacterial defense against inhibitory host factors inherent to different body sites. Two well-characterized multidrug resistance efflux systems (MtrCDE and FarAB-MtrE) exist in Neisseria gonorrhoeae, a bacterial pathogen of the human genital mucosae. In vitro studies suggest that the MtrCDE and FarAB-MtrE efflux systems protect the gonococcus from hydrophobic antimicrobial substances that are likely to be present on mucosal surfaces. Here we report that a functional MtrCDE efflux system, but not a functional FarAB-MtrE system, enhances experimental gonococcal genital tract infection in female mice. Specifically, the recovery of mtrD and mtrE mutants, but not a farB mutant, from mice inoculated with mutant or wild-type gonococci was reduced compared with that of the wild-type strain. Competitive-infection experiments confirmed the survival disadvantage of MtrCDE-deficient gonococci. This report is the first direct evidence that a multidrug resistance efflux system enhances survival of a bacterial pathogen in the genital tract. Additionally, experiments using ovariectomized mice showed that MtrCDE-deficient gonococci were more rapidly cleared from mice that were capable of secreting gonadal hormones. MtrCDE-deficient gonococci were more sensitive to nonphysiological concentrations of progesterone in vitro than were wild-type or FarAB-MtrE-deficient gonococci. These results suggest that progesterone may play an inhibitory role in vivo. However, hormonally regulated factors rather than progesterone itself may be responsible for the more rapid clearance of mtr-deficient gonococci from intact mice.

Multiple gonococcal pilin antigenic variants are produced during experimental human infections.

Gonococcal pilin variation is thought to allow immune evasion and change the adherence properties of the pilus. We have examined the process of pilin antigenic variation in human volunteers inoculated with strain FA1090. Our data show that pilin variation occurred throughout the process of infection, that at each time sampled after inoculation multiple pilin variants were present, and that later pilin variants appear to be recombinants between previously expressed genes and the silent storage pilin copies. Thus, during infection a large repertoire of proteins are available to the population to help avoid immune responses, to provide pili with varying functions, and to transmit to a new host.

Clinically relevant mutations that cause derepression of the Neisseria gonorrhoeae MtrC-MtrD-MtrE Efflux pump system confer different levels of antimicrobial resistance and in vivo fitness

The MtrC‐MtrD‐MtrE efflux pump system confers resistance to macrolide antibiotics and antimicrobial substances of the host innate defence. Clinical isolates with increased resistance to erythromycin and azithromycin frequently harbour mutations in the mtrR structural gene, which encodes a repressor of the mtrCDE operon, or the mtrR promoter region. The MtrC‐MtrD‐MtrE system is important for gonococcal survival in the murine genital tract, and derepression of the mtrCDE operon via deletion of mtrR confers increased fitness in vivo. Here we compared isogenic strains with naturally occurring mtrR locus mutations for differences in mtrCDE expression and pump‐related phenotypes. Mutations upstream of mtrC, including those within the MtrR binding region and a novel mutation that increases mtrC RNA stability conferred the highest levels of derepression as measured by mtrCDE transcription and resistance to antibiotics, progesterone and antimicrobial peptides. In contrast, mutations within the mtrR coding sequence conferred low to intermediate levels of derepression. In vivo, the mtr mutants were more fit than the wild‐type strain, the degree to which paralleled in vitro resistance gradients. These studies establish a hierarchy of mtrR locus mutations with regard to regulation of pump efflux, and suggest selection for more derepressed mutants may occur during mixed infections.

Critical role of Th17 responses in a murine model of Neisseria gonorrhoeae genital infection

Host immune responses, including the characteristic influx of neutrophils, against Neisseria gonorrhoeae are poorly understood; adaptive immunity is minimal and non-protective. We hypothesize that N. gonorrhoeae selectively elicits Th17-dependent responses, which trigger innate defense mechanisms, including neutrophils and antimicrobial proteins, that it can resist. We found that N. gonorrhoeae induced the production of interleukin-17 (IL-17) in mouse T-cells and Th17-inducing cytokines in mouse and human APCs in vitro. IL-17 was induced in the iliac lymph nodes in vivo in a female mouse model of genital tract gonococcal infection. Antibody blockade of IL-17 or deletion of the major IL-17 receptor (IL-17R) in IL-17RAKO mice led to prolonged infection and diminished neutrophil influx. Genital tract tissue from IL-17RAKO mice showed reduced production of neutrophil-attractant chemokines in response to culture with N. gonorrhoeae. These results imply a crucial role for IL-17 and Th17 cells in the immune response to N. gonorrhoeae.

Regulation of the MtrC-MtrD-MtrE Efflux-Pump System Modulates the In Vivo Fitness of Neisseria gonorrhoeae

The Neisseria gonorrhoeae MtrC-MtrD-MtrE multidrug-resistance efflux pump expels macrolide antibiotics, penicillin, and antimicrobial effectors of the innate defense. Mutation of the mtrR locus, which encodes a transcriptional repressor of the mtrCDE operon, increases gonococcal resistance to these agents. Here we report that, in a mouse infection model, an mtrR mutant is more fit than the wild-type bacteria. Consistent with derepression of the mtrCDE operon as the primary reason for the fitness benefit, an mtrR,mtrE double mutant and an mtrE mutant showed no difference in survival phenotype. Gonococcal mutants deficient in MtrA, an activator of the mtrCDE operon, exhibited significantly reduced fitness in vivo, and mtrA mutants with spontaneous compensatory mtrR mutations were selected during infection. These results confirm the importance of the MtrC-MtrD-MtrE efflux-pump system during experimental gonococcal genital-tract infection and also illustrate an antibiotic-resistance mechanism that is accompanied by a fitness benefit rather than a fitness cost.

Tests of Buffergel for contraception and prevention of sexually transmitted diseases in animal models.

Background BufferGel is a novel spermicidal and microbicidal gelformulated to maintain the natural protective acidity of the vagina byacidifying semen, which otherwise alkalinizes thevagina. Goal To test the efficacy of BufferGel for preventing sexuallytransmitted infections and pregnancy in animalmodels. Study Design Animals were challenged with pathogens or sperm afterpretreatment with both test and control agents, or after no pretreatment, thenevaluated for infection or pregnancy using standardmethods. Results BufferGel provided significant contraceptive efficacy inthe rabbit, and significant protection against vaginal and rectal transmissionof herpes simplex virus type 2 (HSV-2) in the mouse, vaginal transmission of Chlamydia trachomatis in the mouse,and skin transmission of cottontail rabbit papillomavirus in the rabbit. Itdid not protect against vaginal transmission of Neisseria gonorrhoeae in themouse. Conclusions The protective efficacy of BufferGel in five of the sixanimal models suggests that this microbicide warrants clinical evaluation forboth contraception and diseaseprevention.

Estradiol-Treated Female Mice as Surrogate Hosts for Neisseria gonorrhoeae Genital Tract Infections

Historically, animal modeling of gonorrhea has been hampered by the exclusive adaptation of Neisseria gonorrhoeae to humans. Genital tract infection can be established in female mice that are treated with 17β-estradiol, however, and many features of experimental murine infection mimic human infection. Here we review the colonization kinetics and host response to experimental murine gonococcal infection, including mouse strain differences and evidence that IL-17 responses, toll-like receptor 4, and T regulatory cells play a role in infection. We also discuss the strengths and limitations of the mouse system and the potential of transgenic mice to circumvent host restrictions. Additionally, we review studies with genetically defined mutants that demonstrated a role for sialyltransferase and the MtrC–MtrD–MtrE active efflux pump in evading innate defenses in vivo, but not for factors hypothesized to protect against the phagocytic respiratory burst and H2O2-producing lactobacilli. Studies using estradiol-treated mice have also revealed the existence of non-host-restricted iron sources in the female genital tract and the influence of hormonal factors on colonization kinetics and selection for opacity (Opa) protein expression. Recent work by others with estradiol-treated mice that are transgenic for human carcinoembryonic adhesion molecules (CEACAMs) supports a role for Opa proteins in enhancing cellular attachment and thus reduced shedding of N. gonorrhoeae. Finally we discuss the use of the mouse model in product testing and a recently developed gonorrhea chlamydia coinfection model.

Local and humoral immune responses against primary and repeat Neisseria gonorrhoeae genital tract infections of 17β-estradiol-treated mice

The 17beta-estradiol-treated mouse model is the only small animal model of gonococcal genital tract infection. Here we show gonococci localized within vaginal and cervical tissue, including the lamina propria, and high numbers of neutrophils and macrophages in genital tissue from infected mice. Infection did not induce a substantial or sustained increase in total or gonococcal-specific antibodies. Mice could be reinfected with the same strain and repeat infection did not boost the antibody response. However, intravaginal immunization of estradiol-treated mice induced gonococcal-specific primary and secondary serum antibody responses. We conclude that similar to human infection, experimental murine infection induces local inflammation but not an acquired immune response or immunological memory.

Experimental Gonococcal Infection in Male Volunteers: Cumulative Experience with Neisseria gonorrhoeae Strains FA1090 and MS11mkC.

Experimental infection of male volunteers with Neisseria gonorrhoeae is safe and reproduces the clinical features of naturally acquired gonococcal urethritis. Human inoculation studies have helped define the natural history of experimental infection with two well-characterized strains of N. gonorrhoeae, FA1090 and MS11mkC. The human model has proved useful for testing the importance of putative gonococcal virulence factors for urethral infection in men. Studies with isogenic mutants have improved our understanding of the requirements for gonococcal LOS structures, pili, opacity proteins, IgA1 protease, and the ability of infecting organisms to obtain iron from human transferrin and lactoferrin during uncomplicated urethritis. The model also presents opportunities to examine innate host immune responses that may be exploited or improved in development and testing of gonococcal vaccines. Here we review results to date with human experimental gonorrhea.

Vaccines against gonorrhea: Current status and future challenges

Gonorrhea occurs at high incidence throughout the world and significantly impacts reproductive health and the spread of human immunodeficiency virus. Current control measures are inadequate and seriously threatened by the rapid emergence of antibiotic resistance. Progress on gonorrhea vaccines has been slow; however, recent advances justify significant effort in this area. Conserved vaccine antigens have been identified that elicit bactericidal antibodies and, or play key roles in pathogenesis that could be targeted by a vaccine-induced response. A murine genital tract infection model is available for systematic testing of antigens, immunization routes and adjuvants, and transgenic mice exist to relieve some host restrictions. Furthermore, mechanisms by which Neisseria gonorrhoeae avoids inducing a protective adaptive response are being elucidated using human cells and the mouse model. Induction of a Th1 response in mice clears infection and induces a memory response, which suggests Th1-inducing adjuvants may be key in vaccine-induced protection. Continued research in this area should include human testing and clinical studies to confirm or negate findings from experimental systems and to define protective host factors.