Pertactin contributes to shedding and transmission of Bordetella bronchiseptica.

Whooping cough is resurging in the United States despite high vaccine coverage. The rapid rise of Bordetella pertussis isolates lacking pertactin (PRN), a key vaccine antigen, has led to concerns about vaccine-driven evolution. Previous studies showed that pertactin can mediate binding to mammalian cells in vitro and act as an immunomodulatory factor in resisting neutrophil-mediated clearance. To further investigate the role of PRN in vivo, we examined the functions of pertactin in the context of a more naturally low dose inoculation experimental system using C3H/HeJ mice that is more sensitive to effects on colonization, growth and spread within the respiratory tract, as well as an experimental approach to measure shedding and transmission between hosts. A B. bronchiseptica pertactin deletion mutant was found to behave similarly to its wild-type (WT) parental strain in colonization of the nasal cavity, trachea, and lungs of mice. However, the pertactin-deficient strain was shed from the nares of mice in much lower numbers, resulting in a significantly lower rate of transmission between hosts. Histological examination of respiratory epithelia revealed that pertactin-deficient bacteria induced substantially less inflammation and mucus accumulation than the WT strain and in vitro assays verified the effect of PRN on the induction of TNF-α by murine macrophages. Interestingly, only WT B. bronchiseptica could be recovered from the spleen of infected mice and were further observed to be intracellular among isolated splenocytes, indicating that pertactin contributes to systemic dissemination involving intracellular survival. These results suggest that pertactin can mediate interactions with immune cells and augments inflammation that contributes to bacterial shedding and transmission between hosts. Understanding the relative contributions of various factors to inflammation, mucus production, shedding and transmission will guide novel strategies to interfere with the reemergence of pertussis.

Pertactin-Deficient Bordetella pertussis, Vaccine-Driven Evolution, and Reemergence of Pertussis.

Recent reemergence of pertussis (whooping cough) in highly vaccinated populations and rapid expansion of Bordetella pertussis strains lacking pertactin (PRN), a common acellular vaccine antigen, have raised the specter of vaccine-driven evolution and potential return of what was once the major killer of children. The discovery that most circulating B. pertussis strains in the United States have acquired new and independent disruptive mutations in PRN is compelling evidence of strong selective pressure. However, the other 4 antigens included in acellular vaccines do not appear to be selected against so rapidly. We consider 3 aspects of PRN that distinguish it from other vaccine antigens, which might, individually or collectively, explain why only this antigen is being precipitously eliminated. An understanding of the increase in PRN-deficient strains should provide useful information for the current search for new protective antigens and provide broader lessons for the design of improved subunit vaccines.

Modeling Immune Evasion and Vaccine Limitations by Targeted Nasopharyngeal Bordetella pertussis Inoculation in Mice.

Conventional pertussis animal models deliver hundreds of thousands of Bordetella pertussis bacteria deep into the lungs, rapidly inducing severe pneumonic pathology and a robust immune response. However, human infections usually begin with colonization and growth in the upper respiratory tract. We inoculated only the nasopharynx of mice to explore the course of infection in a more natural exposure model. Nasopharyngeal colonization resulted in robust growth in the upper respiratory tract but elicited little immune response, enabling prolonged and persistent infection. Immunization with human acellular pertussis vaccine, which prevents severe lung infections in the conventional pneumonic infection model, had little effect on nasopharyngeal colonization. Our infection model revealed that B. pertussis can efficiently colonize the mouse nasopharynx, grow and spread within and between respiratory organs, evade robust host immunity, and persist for months. This experimental approach can measure aspects of the infection processes not observed in the conventional pneumonic infection model.

Generating enhanced mucosal immunity against Bordetella pertussis: current challenges and new directions.

Bordetella pertussis (Bp) is the highly transmissible etiologic agent of pertussis, a severe respiratory disease that causes particularly high morbidity and mortality in infants and young children. Commonly known as "whooping cough," pertussis is one of the least controlled vaccine-preventable diseases worldwide with several countries experiencing recent periods of resurgence despite broad immunization coverage. While current acellular vaccines prevent severe disease in most cases, the immunity they confer wanes rapidly and does not prevent sub clinical infection or transmission of the bacterium to new and vulnerable hosts. The recent resurgence has prompted new efforts to generate robust immunity to Bp in the upper respiratory mucosa, from which colonization and transmission originate. Problematically, these initiatives have been partially hindered by research limitations in both human and animal models as well as potent immunomodulation by Bp. Here, we consider our incomplete understanding of the complex host-pathogen dynamics occurring in the upper airway to propose new directions and methods that may address critical gaps in research. We also consider recent evidence that supports the development of novel vaccines specifically designed to generate robust mucosal immune responses capable of limiting upper respiratory colonization to finally halt the ongoing circulation of Bordetella pertussis.

Novel murine model reveals an early role for pertussis toxin in disrupting neonatal immunity to Bordetella pertussis.

The increased susceptibility of neonates to specific pathogens has previously been attributed to an underdeveloped immune system. More recent data suggest neonates have effective protection against most pathogens but are particularly susceptible to those that target immune functions specific to neonates. Bordetella pertussis (Bp), the causative agent of "whooping cough", causes more serious disease in infants attributed to its production of pertussis toxin (PTx), although the neonate-specific immune functions it targets remain unknown. Problematically, the rapid development of adult immunity in mice has confounded our ability to study interactions of the neonatal immune system and its components, such as virtual memory T cells which are prominent prior to the maturation of the thymus. Here, we examine the rapid change in susceptibility of young mice and define a period from five- to eight-days-old during which mice are much more susceptible to Bp than mice even a couple days older. These more narrowly defined "neonatal" mice display significantly increased susceptibility to wild type Bp but very rapidly and effectively respond to and control Bp lacking PTx, more rapidly even than adult mice. Thus, PTx efficiently blocks some very effective form(s) of neonatal protective immunity, potentially providing a tool to better understand the neonatal immune system. The rapid clearance of the PTx mutant correlates with the early accumulation of neutrophils and T cells and suggests a role for PTx in disrupting their accumulation. These results demonstrate a striking age-dependent response to Bp, define an early age of extreme susceptibility to Bp, and demonstrate that the neonatal response can be more efficient than the adult response in eliminating bacteria from the lungs, but these neonatal functions are substantially blocked by PTx. This refined definition of "neonatal" mice may be useful in the study of other pathogens that primarily infect neonates, and PTx may prove a particularly valuable tool for probing the poorly understood neonatal immune system.

Adaptive immune protection of the middle ears differs from that of the respiratory tract.

The efficacy of the adaptive immune system in the middle ear (ME) is well established, but the mechanisms are not as well defined as those of gastrointestinal or respiratory tracts. While cellular elements of the adaptive response have been detected in the MEs following infections (or intranasal immunizations), their specific contributions to protecting the organ against reinfections are unknown. How immune protection mechanisms of the MEs compares with those in the adjacent and attached upper and lower respiratory airways remains unclear. To address these knowledge gaps, we used an established mouse respiratory infection model that we recently showed also involves ME infections. Bordetella bronchiseptica delivered to the external nares of mice in tiny numbers very efficiently infects the respiratory tract and ascends the Eustachian tube to colonize and infect the MEs, where it causes severe but acute inflammation resembling human acute otitis media (AOM). Since this AOM naturally resolves, we here examine the immunological mechanisms that clear infection and protect against subsequent infection, to guide efforts to induce protective immunity in the ME. Our results show that once the MEs are cleared of a primary B. bronchiseptica infection, the convalescent organ is strongly protected from reinfection by the pathogen despite its persistence in the upper respiratory tract, suggesting important immunological differences in these adjacent and connected organs. CD4+ and CD8+ T cells trafficked to the MEs following infection and were necessary to robustly protect against secondary challenge. Intranasal vaccination with heat killed B. bronchiseptica conferred robust protection against infection to the MEs, even though the nasopharynx itself was only partially protected. These data establish the MEs as discrete effector sites of adaptive immunity and shows that effective protection in the MEs and the respiratory tract is significantly different. This model system allows the dissection of immunological mechanisms that can prevent bacteria in the nasopharynx from ascending the ET to colonize the ME.

Natural History and Ecology of Interactions Between Bordetella Species and Amoeba.

A variety of bacteria have evolved the ability to interact with environmental phagocytic predators such as amoebae, which may have facilitated their subsequent interactions with phagocytes in animal hosts. Our recent study found that the animal pathogen Bordetella bronchiseptica can evade predation by the common soil amoeba Dictyostelium discoideum, survive within, and hijack its complex life cycle as a propagation and dissemination vector. However, it is uncertain whether the mechanisms allowing interactions with predatory amoebae are conserved among Bordetella species, because divergence, evolution, and adaptation to different hosts and ecological niches was accompanied by acquisition and loss of many genes. Here we tested 9 diverse Bordetella species in three assays representing distinct aspects of their interactions with D. discoideum. Several human and animal pathogens retained the abilities to survive within single-celled amoeba, to inhibit amoebic plaque expansion, and to translocate with amoebae to the fruiting body and disseminate along with the fruiting body. In contrast, these abilities were partly degraded for the bird pathogen B. avium, and for the human-restricted species B. pertussis and B. parapertussis. Interestingly, a different lineage of B. parapertussis only known to infect sheep retained the ability to interact with D. discoideum, demonstrating that these abilities were lost in multiple lineages independently, correlating with niche specialization and recent rapid genome decay apparently mediated by insertion sequences. B. petrii has been isolated sporadically from diverse human and environmental sources, has acquired insertion sequences, undergone genome decay and has also lost the ability to interact with amoebae, suggesting some specialization to some unknown niche. A genome-wide association study (GWAS) identified a set of genes that are potentially associated with the ability to interact with D. discoideum. These results suggest that massive gene loss associated with specialization of some Bordetella species to a closed life cycle in a particular host was repeatedly and independently accompanied by loss of the ability to interact with amoebae in an environmental niche.

Modeling the catarrhal stage of Bordetella pertussis upper respiratory tract infections in mice.

Pertussis (whooping cough) is a highly transmissible human respiratory disease caused by Bordetella pertussis, a human-restricted pathogen. Animal models generally involve pneumonic infections induced by depositing large numbers of bacteria in the lungs of mice. These models have informed us about the molecular pathogenesis of pertussis and guided development of vaccines that successfully protect against severe disease. However, they bypass the catarrhal stage of the disease, when bacteria first colonize and initially grow in the upper respiratory tract. This is a critical and highly transmissible stage of the infection that current vaccines do not prevent. Here, we demonstrate a model system in which B. pertussis robustly and persistently infects the nasopharynx of TLR4-deficient mice, inducing localized inflammation, neutrophil recruitment and mucus production as well as persistent shedding and occasional transmission to cage mates. This novel experimental system will allow the study of the contributions of bacterial factors to colonization of and shedding from the nasopharynx, as occurs during the catarrhal stage of pertussis, and interventions that might better control the ongoing circulation of pertussis.

Probing Immune-Mediated Clearance of Acute Middle Ear Infection in Mice.

Acute otitis media (AOM) is commonly caused by bacterial pathobionts of the nasopharynx that ascend the Eustachian tube to cause disease in the middle ears. To model and study the various complexities of AOM, common human otopathogens are injected directly into the middle ear bullae of rodents or are delivered with viral co-infections which contribute to the access to the middle ears in complex and partially understood ways. Here, we present the novel observation that Bordetella bronchiseptica, a well-characterized respiratory commensal/pathogen of mice, also efficiently ascends their Eustachian tubes to colonize their middle ears, providing a flexible mouse model to study naturally occurring AOM. Mice lacking T and/or B cells failed to resolve infections, highlighting the cooperative role of both in clearing middle ear infection. Adoptively transferred antibodies provided complete protection to the lungs but only partially protected the middle ears, highlighting the differences between respiratory and otoimmunology. We present this as a novel experimental system that can capitalize on the strengths of the mouse model to dissect the molecular mechanisms involved in the generation and function of immunity within the middle ear.