Bordetella parapertussis Survives inside Human Macrophages in Lipid Raft-Enriched Phagosomes

Bordetella parapertussis is a human pathogen that causes whooping cough. The increasing incidence of B. parapertussis has been attributed to the lack of cross protection induced by pertussis vaccines. It was previously shown that B. parapertussis is able to avoid bacterial killing by polymorphonuclear leukocytes (PMN) if specific opsonic antibodies are not present at the site of interaction. Here, we evaluated the outcome of B. parapertussis innate interaction with human macrophages, a less aggressive type of cell and a known reservoir of many persistent pathogens. The results showed that in the absence of opsonins, O antigen allows B. parapertussis to inhibit phagolysosomal fusion and to remain alive inside macrophages. The O antigen targets B. parapertussis to lipid rafts that are retained in the membrane of phagosomes that do not undergo lysosomal maturation. Forty-eight hours after infection, wild-type B. parapertussis bacteria but not the O antigen-deficient mutants were found colocalizing with lipid rafts and alive in nonacidic compartments. Taken together, our data suggest that in the absence of opsonic antibodies, B. parapertussis survives inside macrophages by preventing phagolysosomal maturation in a lipid raft- and O antigen-dependent manner. Two days after infection, about 15% of macrophages were found loaded with live bacteria inside flotillin-enriched phagosomes that had access to nutrients provided by the host cell recycling pathway, suggesting the development of an intracellular infection. IgG opsonization drastically changed this interaction, inducing efficient bacterial killing. These results highlight the need for B. parapertussis opsonic antibodies to induce bacterial clearance and prevent the eventual establishment of cellular reservoirs of this pathogen.Fil: Gorgojo, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Harvill, Eric. State University of Pennsylvania; Estados UnidosFil: Rodriguez, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentin

Bacteriophage-mediated competition in Bordetella bacteria

Apparent competition between species is believed to be one of the principle driving forces that structure ecological communities, although the precise mecha nisms have yet to be characterized. Here we develop a model system that isolates phage-mediated interactions by neutralizing resource competition using two genetically identical Bordetella bronchiseptica strains that differ only in that one is the carrier of a phage and the other is susceptible to the phage. We observe and quantify the competitive advantage of the bacterial strain bearing the prophage in both invading and in resisting invasion by bacteria susceptible to the phage, and use our measurements to develop a mathematical model of phage-mediated competition. The model predicts, and experimental evidence confirms, that the competitive advantage conferred by the phage depends only on the relative phage pathology and is independent of other phage and host parameters. This work combines experimental and mathematical approaches to the study of phage-driven competition, and provides an experimentally tested framework for evaluation of the effects of pathogens/parasites on interspecific competition.Comment: 10pages, 8 figure

Bordetella parapertussis survives inside human macrophages in lipid raft-enriched phagosomes

Bordetella parapertussis is a human pathogen that causes whooping cough. The increasing incidence of B. parapertussis has been attributed to the lack of cross protection induced by pertussis vaccines. It was previously shown that B. parapertussis is able to avoid bacterial killing by polymorphonuclear leukocytes (PMN) if specific opsonic antibodies are not present at the site of interaction. Here, we evaluated the outcome of B. parapertussis innate interaction with human macrophages, a less aggressive type of cell and a known reservoir of many persistent pathogens. The results showed that in the absence of opsonins, O antigen allows B. parapertussis to inhibit phagolysosomal fusion and to remain alive inside macrophages. The O antigen targets B. parapertussis to lipid rafts that are retained in the membrane of phagosomes that do not undergo lysosomal maturation. Fortyeight hours after infection, wild-type B. parapertussis bacteria but not the O antigen-deficient mutants were found colocalizing with lipid rafts and alive in nonacidic compartments. Taken together, our data suggest that in the absence of opsonic antibodies, B. parapertussis survives inside macrophages by preventing phagolysosomal maturation in a lipid raft- and O antigen-dependent manner. Two days after infection, about 15% of macrophages were found loaded with live bacteria inside flotillin-enriched phagosomes that had access to nutrients provided by the host cell recycling pathway, suggesting the development of an intracellular infection. IgG opsonization drastically changed this interaction, inducing efficient bacterial killing. These results highlight the need for B. parapertussis opsonic antibodies to induce bacterial clearance and prevent the eventual establishment of cellular reservoirs of this pathogen.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Maternal vaccination: shaping the neonatal response to pertussis

Antepartum maternal vaccination can protect highly sensitive newborns before they are old enough to receive their own vaccines. Two vaccines are currently recommended during pregnancy: the flu vaccine and the Tdap vaccine against tetanus, diphtheria, and pertussis. Although there is strong evidence that maternal vaccination works to protect the offspring, limitations in the understanding of vaccines and of maternal transfer of immunity compound to obscure our understanding of how they work. Here we focus on the example of pertussis to explore the possible mechanisms involved in the transfer of protection to offspring and how these may impact the newborn’s response to future exposure to pertussis. For example, Tdap vaccines induce pathogen specific antibodies, and those antibodies are known to be transferred from mother to the fetus in utero and to the newborn via milk. But antibodies alone have modest impact on pertussis disease, and even less effect on colonization/transmission. Maternal immune cells can also be transferred to offspring and may play a direct role in protection from disease and/or influence the developing neonatal immune system. However, some of the transferred immunity may also blunt the offspring’s response to subsequent vaccination. In this review we will summarize the protection conferred to offspring by maternal vaccination against pertussis and the likely mechanisms by which protection is transferred, identifying the many knowledge gaps that limit our most effective application of this approach

A Newly Discovered Bordetella Species Carries a Transcriptionally Active CRISPR-Cas with a Small Cas9 Endonuclease

Background Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. Methods The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Results Here we describe a novel Type II-C CRISPR and its associated genes—cas1, cas2, and cas9—in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Conclusions Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies

The O antigen is a critical antigen for the development of a protective immune response to Bordetella parapertussis

Despite excellent vaccine coverage in developed countries, whooping cough is a reemerging disease that can be caused by two closely related pathogens, Bordetella pertussis and B. parapertussis. The two are antigenically distinct, and current vaccines, containing only B. pertussis-derived antigens, confer efficient protection against B. pertussis but not against B. parapertussis. B. pertussis does not express the O antigen, while B. parapertussis retains it as a dominant surface antigen. Since the O antigen is a protective antigen for many pathogenic bacteria, we examined whether this factor is a potential protective antigen for B. parapertussis. In a mouse model of infection, immunization with wild-type B. parapertussis elicited a strong antibody response to the O antigen and conferred efficient protection against a subsequent B. parapertussis challenge. However, immunization with an isogenic mutant lacking the O antigen, B. parapertussis Δwbm, induced antibodies that recognized other antigens but did not efficiently mediate opsonophagocytosis of B. parapertussis. The passive transfer of sera raised against B. parapertussis, but not B. parapertussis Δwbm, reduced B. parapertussis loads in the lower respiratory tracts of mice. The addition of 10 μg of purified B. parapertussis lipopolysaccharide (LPS), which contains the O antigen, but not B. parapertussis Δwbm LPS drastically improved the efficacy of the acellular vaccine Adacel against B. parapertussis. These data suggest that the O antigen is a critical protective antigen of B. parapertussis and its inclusion can substantially improve whooping cough vaccine efficacy against this pathogen.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Molecular signature of hypersaline adaptation: insights from genome and proteome composition of halophilic prokaryotes

A comparative genomic and proteomic study of halophilic and non-halophilic prokaryotes identifies specific genomic and proteomic features typical of halophilic species that are independent from genomic GC-content and taxonomic position

Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms

B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through nonphagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts.Centro de Investigación y Desarrollo en Fermentaciones IndustrialesInstituto de Biotecnologia y Biologia Molecula

Modeling Systems-Level Regulation of Host Immune Responses

Many pathogens are able to manipulate the signaling pathways responsible for the generation of host immune responses. Here we examine and model a respiratory infection system in which disruption of host immune functions or of bacterial factors changes the dynamics of the infection. We synthesize the network of interactions between host immune components and two closely related bacteria in the genus Bordetellae. We incorporate existing experimental information on the timing of immune regulatory events into a discrete dynamic model, and verify the model by comparing the effects of simulated disruptions to the experimental outcome of knockout mutations. Our model indicates that the infection time course of both Bordetellae can be separated into three distinct phases based on the most active immune processes. We compare and discuss the effect of the species-specific virulence factors on disrupting the immune response during their infection of naive, antibody-treated, diseased, or convalescent hosts. Our model offers predictions regarding cytokine regulation, key immune components, and clearance of secondary infections; we experimentally validate two of these predictions. This type of modeling provides new insights into the virulence, pathogenesis, and host adaptation of disease-causing microorganisms and allows systems-level analysis that is not always possible using traditional methods