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Mechanisms of Bacterial Virulence and Disease Prevention

Research Summary

Andrew Camilli is interested in understanding how bacterial pathogens cause disease, establish environmental reservoirs, and are transmitted. In addition he is interested in developing vaccines and phage products to prevent disease.

Our laboratory investigates bacterial determinants that are important in the virulence, transmission, and dissemination of mucosal pathogens. We use Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, as a model intestinal pathogen. V. cholerae is water-borne and maintains a reservoir in fresh and salt water in endemic areas. We are investigating the roles of V. cholerae genes at each stage of its life cycle, including both environmental and infectious stages. In addition, we study Streptococcus pneumoniae (pneumococcus) as a model respiratory tract pathogen. Pneumococcus is a commensal of the human nasopharynx, but can cause deadly invasive disease when it spreads into the lung or bloodstream. We found that pneumococcus can survive for long periods in a desiccated state on environmental surfaces, which may serve as a source of transmission.

Determining the Roles of Genes at Different Stages of the Pathogen Life Cycle
Despite many decades of study we still lack knowledge of the function and expression patterns of most genes in the V. cholerae and pneumococcus genomes. This makes understanding many aspects of their transmission, pathogenesis, and dissemination difficult, and hinders the development of vaccines, antibiotics, and diagnostics. We are developing genetic tools to increase knowledge of the roles of pathogen genes at each stage of their life cycles. One such tool for determining patterns of gene expression, is recombination-based in vivo expression technology (RIVET). This method uses a site-specific DNA recombinase as a transcriptional reporter. When expressed, the recombinase mediates the excision and loss of a selectable marker from the genome. In this way, gene induction is coupled to a heritable phenotypic change, allowing us to know if, when, and where a gene is turned on. Using RIVET we determined the complex temporal pattern of virulence and environmental gene expression that occurs when V. cholerae enters into and out of the host intestine. Through this work, we discovered that the cyclic dinucleotide c-di-GMP functions as an important second messenger in V. cholerae to mediate the inverse regulation of virulence and environmental survival genes.

Another genetic tool we developed is transposon-sequencing (Tn-seq), which combines saturating transposon mutagenesis with next-generation sequencing of the transposon-genome junctions. By counting the number of reads corresponding to each transposon insertion within a library of mutants, we can measure changes in mutant frequency, thus allowing us to quantify the contribution of genes to phenotypes of interest. Tn-seq also reveals which genes are essential for viability, since insertions in such genes are absent from an otherwise saturating library. Essential genes represent ideal targets for vaccine or antimicrobial drug development. Because of the depth and scale of next-generation sequencing, Tn-seq is extremely accurate and sensitive, revealing even minute changes in fitness, which in turn increases our knowledge of genes that contribute to important processes. We are using Tn-seq on V. cholerae and pneumococcus in numerous conditions, including animal models of infection, in order to determine the contributions of each of their genes. For V. cholerae, we have developed novel models of transmission and dissemination to aid these studies. Using these assays, we uncovered roles for many known and hypothetical genes. For pneumococcus, we performed Tn-seq in models of nasopharyngeal carriage and lung infection, and in addition, in a large number of in vitro conditions that mimic certain aspects of host infection such as stresses and growth on individual carbon sources. By overlaying these data sets, we obtained the most detailed gene-condition map yet of a bacterial pathogen (Figure 1).

Vaccine Development and Mechanisms of Immunity
Taking advantage of a natural process of gram-negative bacteria — the shedding of outer membrane vesicles (OMVs) — we developed a novel cholera vaccine composed of purified OMVs. We showed that OMVs administered orally or intranasally result in a robust, long-lasting mucosal immune response, which protects from challenge. In characterizing this immune response, we discovered that antibodies recognizing the lipopolysaccharide (LPS) of V. cholerae were necessary and sufficient for protection. Furthermore, we showed that the mechanism of protection is blocking of bacterial motility, which may be the same for other types of cholera vaccines and of natural immunity.

Although capsular polysaccharide conjugate vaccines against pneumococcus are highly effective, they are too expensive for use in most of the world, and only protect from a fraction of capsular types. An alternative vaccine strategy is to use highly conserved surface protein antigens, which could theoretically provide protection from any capsular type. Using knowledge of gene function from projects described above, we are developing two types of protein-based pneumococcal vaccines: an acapsular live-attenuated strain, and a multi-subunit acellular vaccine. For the former, we are deleting important transcriptional regulators in order to engineer pneumococcus to constitutively express many antigenic surface proteins. This serves to attenuate virulence and increase presentation of protective antigens to the host immune system. For the latter type of vaccine, we are using Tn-seq to identify the most important surface proteins. We are then testing combinations of those that are highly conserved, for generating a protective and long-lasting immune response.

Impact of Lytic Phages on the V. cholerae Life Cycle
Lytic phages are bactericidal viruses that exist in dynamic equilibrium with their hosts. We are isolating and characterizing lytic phages frequently found in stools of cholera patients. Through studying the evolutionary arms race between these phages and V. cholerae we identified a novel type of chromosomal element called a PICI (phage-inducible chromosomal island)-like element (PLE). The PLE protects V. cholerae from one very common type of phage. We subsequently discovered that a variant of this phage has, in turn, acquired a CRISPR/Cas system that it uses to inactivate the PLE, thus giving the phage the upper hand (Figure 2). Through this project, we have obtained conclusive evidence that lytic phages are imposing strong selective pressures on V. cholerae within the context of human infection, which in turn is shaping the genotype and phenotype of the shed bacteria. We are using this knowledge to develop phage cocktails that can be used to prevent cholera.

Grants from the National Institutes of Health, The Pew Charitable Trusts, and the Bill & Melinda Gates Foundation provided partial support for these projects.

As of April 24, 2015

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Tufts University School of Medicine