Eric J. Nelson

Cholera transmission: the host, pathogen and bacteriophage dynamic

Zimbabwe offers the most recent example of the tragedy that befalls a country and its people when cholera strikes. The 2008–2009 outbreak rapidly spread across every province and brought rates of mortality similar to those witnessed as a consequence of cholera infections a hundred years ago. In this Review we highlight the advances that will help to unravel how interactions between the host, the bacterial pathogen and the lytic bacteriophage might propel and quench cholera outbreaks in endemic settings and in emergent epidemic regions such as Zimbabwe.

Immunization with Vibrio cholerae Outer Membrane Vesicles Induces Protective Immunity in Mice

ABSTRACT The gram-negative bacterium Vibrio cholerae releases outer membrane vesicles (OMVs) during growth. In this study, we immunized female mice by the intranasal, intragastric, or intraperitoneal route with purified OMVs derived from V. cholerae. Independent of the route of immunization, mice induced specific, high-titer immune responses of similar levels against a variety of antigens present in the OMVs. After the last immunization, the half-maximum total immunoglobulin titer was stable over a 3-month period, indicating that the immune response was long lasting. The induction of specific isotypes, however, was dependent on the immunization route. Immunoglobulin A, for example, was induced to a significant level only by mucosal immunization, with the intranasal route generating the highest titers. We challenged the offspring of immunized female mice with V. cholerae via the oral route in two consecutive periods, approximately 30 and 95 days after the last immunization. Regardless of the route of immunization, the offspring was protected against colonization with V. cholerae in both challenge periods. Our results show that mucosal immunizations via both routes with OMVs derived from V. cholerae induce long-term protective immune responses against this gastrointestinal pathogen. These findings may contribute to the development of “nonliving,” OMV-based vaccines against V. cholerae and other enteric pathogens, using the oral or intranasal route of immunization.

Characterization of Vibrio cholerae outer membrane vesicles as a candidate vaccine for cholera.

ABSTRACT Outer membrane vesicles (OMVs) offer a new approach for an effective cholera vaccine. We recently demonstrated that immunization of female mice with OMVs induces a long-lasting immune response and results in protection of their neonatal offspring from Vibrio cholerae intestinal colonization. This study investigates the induced protective immunity observed after immunization with OMVs in more detail. Analysis of the stomach contents and sera of the neonates revealed significant amounts of anti-OMV immunoglobulins (Igs). Swapping of litters born to immunized and nonvaccinated control mice allowed us to distinguish between prenatal and neonatal uptakes of Igs. Transfer of Igs to neonates via milk was sufficient for complete protection of the neonates from colonization with V. cholerae, while prenatal transfer alone reduced colonization only. Detection of IgA and IgG1 in the fecal pellets of intranasally immunized adult mice indicates an induced immune response at the mucosal surface in the gastrointestinal tract, which is the site of colonization by V. cholerae. When a protocol with three intranasal immunizations 14 days apart was used, the OMVs proved to be efficacious at doses as low as 0.025 μg per immunization. This is almost equivalent to OMV concentrations found naturally in the supernatants of LB-grown cultures of V. cholerae. Heterologous expression of the periplasmic alkaline phosphatase (PhoA) of Escherichia coli resulted in the incorporation of PhoA into OMVs derived from V. cholerae. Intranasal immunization with OMVs loaded with PhoA induced a specific immune response against this heterologous antigen in mice. The detection of an immune response against this heterologously expressed protein is a promising step toward the potential use of OMVs as antigen delivery vehicles in vaccine design.

Cholera stool bacteria repress chemotaxis to increase infectivity

Factors that enhance the transmission of pathogens are poorly understood. We show that Vibrio cholerae shed in human ‘rice‐water’ stools have a 10‐fold lower oral infectious dose in an animal model than in vitro grown V. cholerae, which may aid in transmission during outbreaks. Furthermore, we identify a bacterial factor contributing to this enhanced infectivity: The achievement of a transient motile but chemotaxis‐defective state upon shedding from humans. Rice‐water stool V. cholerae have reduced levels of CheW‐1, which is essential for chemotaxis, and were consequently shown to have a chemotaxis defect when tested in capillary assays. Through mutational analyses, such a state is known to enhance the infectivity of V. cholerae. This is the first report of a pathogen altering its chemotactic state in response to human infection in order to enhance its transmission.

Transmission of Vibrio cholerae Is Antagonized by Lytic Phage and Entry into the Aquatic Environment

Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable) because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water—the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment.

Complexity of rice-water stool from patients with Vibrio cholerae plays a role in the transmission of infectious diarrhea

At the International Centre for Diarrhoeal Disease Research, Bangladesh, one-half of the rice-water stool samples that were culture-positive for Vibrio cholerae did not contain motile V. cholerae by standard darkfield microscopy and were defined as darkfield-negative (DF−). We evaluated the host and microbial factors associated with DF status, as well as the impact of DF status on transmission. Viable counts of V. cholerae in DF− stools were three logs lower than in DF+ stools, although DF− and DF+ stools had similar direct counts of V. cholerae by microscopy. In DF− samples, non-V. cholerae bacteria outnumbered V. cholerae 10:1. Lytic V. cholerae bacteriophage were present in 90% of DF− samples compared with 35% of DF+ samples, suggesting that bacteriophage may limit culture-positive patients from producing DF+ stools. V. cholerae in DF− and DF+ samples were found both planktonically and in distinct nonplanktonic populations; the distribution of organisms between these compartments did not differ appreciably between DF− and DF+ stools. This biology may impact transmission because epidemiological data suggested that household contacts of a DF+ index case were at greater risk of infection with V. cholerae. We propose a model in which V. cholerae multiply in the small intestine to produce a fluid niche that is dominated by V. cholerae. If lytic phage are present, viable counts of V. cholerae drop, stools become DF−, other microorganisms bloom, and cholera transmission is reduced.

A Novel lux Operon in the Cryptically Bioluminescent Fish Pathogen Vibrio salmonicida Is Associated with Virulence

ABSTRACT The cold-water-fish pathogen Vibrio salmonicida expresses a functional bacterial luciferase but produces insufficient levels of its aliphatic-aldehyde substrate to be detectably luminous in culture. Our goals were to (i) better explain this cryptic bioluminescence phenotype through molecular characterization of the lux operon and (ii) test whether the bioluminescence gene cluster is associated with virulence. Cloning and sequencing of the V. salmonicida lux operon revealed that homologs of all of the genes required for luminescence are present: luxAB (luciferase) and luxCDE (aliphatic-aldehyde synthesis). The arrangement and sequence of these structural lux genes are conserved compared to those in related species of luminous bacteria. However, V. salmonicida strains have a novel arrangement and number of homologs of the luxR and luxI quorum-sensing regulatory genes. Reverse transcriptase PCR analysis suggests that this novel arrangement of quorum-sensing genes generates antisense transcripts that may be responsible for the reduced production of bioluminescence. In addition, infection with a strain in which the luxA gene was mutated resulted in a marked delay in mortality among Atlantic salmon relative to infection with the wild-type parent in single-strain challenge experiments. In mixed-strain competition between the luxA mutant and the wild type, the mutant was attenuated up to 50-fold. It remains unclear whether the attenuation results from a direct loss of luciferase or a polar disturbance elsewhere in the lux operon. Nevertheless, these findings document for the first time an association between a mutation in a structural lux gene and virulence, as well as provide a new molecular system to study Vibrio pathogenesis in a natural host.

Atomic geometry of the PbS(100) surface

Abstract The surface structure of PbS(100), in the form of the mineral galena, was studied with the X-ray standing wave technique using surface-sensitive Auger electron yield detection and normal incidence geometry. We find that neither S nor Pb surface atoms are displaced from their respective bulk site positions. This lack of surface relaxation differs from the recent suggestion based on theory that a sizable contraction is needed to explain the experimentally observed lack of a surface core-level shift in Pb 5d photoemission data. To shed more light on this issue, we have also measured high-resolution Pb 4f, Pb 5d, and S 2p photoemission spectra for PbS(100) and confirmed that the surface core-level shifts are absent for cations as well as for anions.

Reaction of water with MgO(100) surfaces: Part III. X-ray standing wave studies

Abstract Clean MgO(100) surfaces cleaved in vacuum and exposed to water vapor or bulk water were studied using the X-ray standing wave (XSW) technique in back reflection mode and surface sensitive, element specific O KLL and Mg KLL Auger electron yield detection. The effects of surface charging were mitigated, but not entirely eliminated, by using a low-energy electron flood gun. Simulation of the XSW signal showed that the effect of surface charging on the XSW data could be minimized with careful experimental design. We demonstrate that the XSW method can be applied to studies of insulating surfaces, and our results for MgO(100) surfaces exposed to water vapor or bulk water indicate the following: (1) the vacuum-cleaved clean surface undergoes no surface reconstruction or significant relaxation perpendicular to the surface; (2) Mg–OH distances on surfaces exposed to water vapor or bulk water measured perpendicular to the (100) surface are the same as in bulk MgO; and (3) the z -position of the surface Mg atoms does not change within the estimated error [±2% of the (200) spacing] after the surface is fully hydroxylated. Our results for the clean, vacuum-cleaved surface disagree with results from impact collision ion-scattering spectroscopy and surface-extended electron-loss fine structure for MgO(100), which indicate 15 and 17% inward relaxation, respectively, and they support results from low-energy electron diffraction, reflection high-energy electron diffraction, and photoelectron diffraction that show little, if any, relaxation or rumpling of the surface.

Photoemission study of Na and Cs adsorption on MgO(100)1 × 1

Abstract Adsorption of Na and Cs on vacuum-cleaved MgO(100)1 × 1 surfaces has been studied with core and valence band photoemission spectroscopy. We conclude that both Na and Cs interact weakly with the surface except for small quantities of the initially adsorbed metals. Only trace amounts (≤ 0.04 monolayers (ML)) of Cs could be found on the surface after doses that exposed the surface to many monolayers of Cs. Although larger amounts of Na (≤ 1 ML) could be condensed on the surface, most of the Na is weakly physisorbed and easily desorbs at room temperature. However, a small fraction (0.02−0.045 ML) of both Na and Cs dosed onto MgO(100) appears to chemisorb on defect sites as indicated by an inability to desorb this fraction. The photoemission spectra for MgO(100) exposed to Na and Cs are unaffected by their presence except at high Na doses, where small changes are observed on the low kinetic energy side of the valence band and the high kinetic energy side of the Mg2p feature.