Moraxella catarrhalis-produced nitric oxide has dual roles in pathogenicity and clearance of infection in bacterial-host cell co-cultures.

In humans, the free radical nitric oxide (NO) is a concentration-dependent multifunctional signaling or toxic molecule that modulates various physiological and pathological processes, and innate immunity against bacterial infections. Because the expression of bacterial genes encoding nitrite reductase (AniA) and NO reductase (NorB) is highly upregulated in biofilms in vitro, it is important to investigate whether bacterial NO-metabolism might subvert host NO signaling and play pathogenic roles during infection. The Moraxella catarrhalis AniA and NorB directly function in production and reduction of NO. Using M. catarrhalis-human bronchial epithelial cell (HBEC) co-cultures, we recently reported AniA/nitrite-dependent cytotoxic effects on HBECs, including altered protein profiles of HBECs and induced HBEC apoptosis, suggesting bacterial nitrite reduction likely dysregulates host cell gene expression. To further clarify whether nitrite reduction-derived NO or nitrite-dependent stimulation of bacterial growth was responsible for adverse effects on HBECs, we monitored bacterial nitrite reduction, levels of NO in co-cultures and resulted dynamic effects on HBEC proliferation and bacterial viability. This study demonstrated that M. catarrhalis nitrite reduction-derived NO was responsible for observed adverse effects on HBECs at mid-to-late stages of infection. More importantly, our data showed that while nitrite promoted bacterial growth and biofilm formation at early hours of infection, nitrite reduction-derived NO was toxic towards M. catarrhalis in maturing biofilms, suggesting nitrite reduction-derived NO might be a possible dualistic mechanism by which M. catarrhalis promotes diseases and spontaneous resolutions.

Human Complement Bactericidal Responses to a Group A Meningococcal Conjugate Vaccine in Africans and Comparison to Responses Measured by 2 Other Group A Immunoassays.

BACKGROUND: PsA-TT (MenAfriVac) is a conjugated polysaccharide vaccine developed to eliminate group A meningococcal disease in Africa. Vaccination of African study participants with 1 dose of PsA-TT led to the production of anti-A polysaccharide antibodies and increased serum bactericidal activity measured using rabbit complement (rSBA). Bactericidal responses measured with human complement (hSBA) are presented here. METHODS: Sera collected before and at 28 days and 1 year after vaccination with either PsA-TT or quadrivalent polysaccharide vaccine (PsACWY) from a random, age-distributed 360-subject subset of the Meningitis Vaccine Project study of PsA-TT in Africans aged 2-29 years were tested for hSBA. Geometric mean titer, fold-rise, and threshold analyses were compared between vaccine groups and age groups. hSBA, rSBA, and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) results were compared and assay correlation and agreement determined. RESULTS: hSBA responses to PsA-TT were substantially higher than those to PsACWY at 28 days and 1 year following immunization, similar to previously reported rSBA and IgG results. The hSBA and IgG ELISA results identified differences between age groups that were not evident by rSBA. The rSBA data indicated sustained high titers 1 year after immunization, whereas hSBA GMTs at 1 year approached 4 in young children. CONCLUSIONS: The high level of protection following PsA-TT immunization campaigns is consistent with the strong hSBA immune responses observed here. Future implementation decisions will likely depend on immunologic data and their long-term correlation with disease and carriage prevention. Expanded immunologic and epidemiologic surveillance may improve the interpretation of differences between these immunoassays.

Development and use of a serum bactericidal assay using pooled human complement to assess responses to a meningococcal group A conjugate vaccine in African toddlers.

A meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused by Neisseria meningitidis. Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC') was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC' and then used the SBA assay with hC' (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12- to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC' lots in two laboratories. hSBA titers of clinical trial sera were then determined using this complement sourcing method. Two different functional antibody tests were necessary for screening hC'. hSBA titers determined using three independent lots of pooled hC' were within expected assay variation among lots and between laboratories. In African toddlers, PsA-TT elicited higher hSBA titers than meningococcal polysaccharide or Hib vaccines. PsA-TT immunization or PS challenge of PsA-TT-primed subjects resulted in vigorous hSBA memory responses, and titers persisted in boosted groups for over a year. Quantifying SBA using pooled hC' is feasible and showed that PsA-TT was highly immunogenic in African toddlers.

Bacterium-generated nitric oxide hijacks host tumor necrosis factor alpha signaling and modulates the host cell cycle in vitro.

In mammalian cells, nitric oxide (NO·) is an important signal molecule with concentration-dependent and often controversial functions of promoting cell survival and inducing cell death. An inducible nitric oxide synthase (iNOS) in various mammalian cells produces higher levels of NO· from l-arginine upon infections to eliminate pathogens. In this study, we reveal novel pathogenic roles of NO· generated by bacteria in bacterium-host cell cocultures using Moraxella catarrhalis, a respiratory tract disease-causing bacterium, as a biological producer of NO·. We recently demonstrated that M. catarrhalis cells that express the nitrite reductase (AniA protein) can produce NO· by reducing nitrite. Our study suggests that, in the presence of pathophysiological levels of nitrite, this opportunistic pathogen hijacks host cell signaling and modulates host gene expression through its ability to produce NO· from nitrite. Bacterium-generated NO· significantly increases the secretion of tumor necrosis factor alpha (TNF-α) and modulates the expression of apoptotic proteins, therefore triggering host cell programmed death partially through TNF-α signaling. Furthermore, our study reveals that bacterium-generated NO· stalls host cell division and directly results in the death of dividing cells by reducing the levels of an essential regulator of cell division. This study provides unique insight into why NO· may exert more severe cytotoxic effects on fast growing cells, providing an important molecular basis for NO·-mediated pathogenesis in infections and possible therapeutic applications of NO·-releasing molecules in tumorigenesis. This study strongly suggests that bacterium-generated NO· can play important pathogenic roles during infections.

Local and humoral immune responses against primary and repeat Neisseria gonorrhoeae genital tract infections of 17beta-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.

Identification of a new OmpA-like protein in Neisseria gonorrhoeae involved in the binding to human epithelial cells and in vivo colonization.

Outer membrane protein As (OmpAs) are highly conserved proteins within the Enterobacteriaceae family. OmpA contributes to the maintenance of structural membrane integrity and invasion into mammalian cells. In Escherichia coli K1 OmpA also contributes to serum resistance and is involved in the virulence of the bacterium. Here we describe the identification of an OmpA-like protein in Neisseria gonorrhoeae (Ng-OmpA). We show that the gonococcal OmpA-like protein, similarly to E. coli OmpA, plays a significant role in the adhesion and invasion into human cervical carcinoma and endometrial cells and is required for entry into macrophages and intracellular survival. Furthermore, the isogenic knockout ompA mutant demonstrates reduced recovery in a mouse model of infection when compared with the wild-type strain, suggesting that Ng-OmpA plays an important role in the in vivo colonization. All together, these data suggest that the newly identified surface exposed protein Ng-OmpA represents a novel virulence factor of gonococcus.