Bordetella pertussiscauses the disease whooping cough through coordinated control of virulence factors by theBordetellavirulence gene system. Microarrays and, more recently, RNA sequencing (RNA-seq) have been used to describein vitrogene expression profiles ofB. pertussisand other pathogens. In previous studies, we have analyzed thein vitrogene expression profiles ofB. pertussis, and we hypothesize that the infection transcriptome profilein vivois significantly different from that under laboratory growth conditions. To study the infection transcriptome ofB. pertussis, we developed a simple filtration technique for isolation of bacteria from infected lungs. The work flow involves filtering the bacteria out of the lung homogenate using a 5-μm-pore-size syringe filter. The captured bacteria are then lysed to isolate RNA for Illumina library preparation and RNA-seq analysis. Upon comparing thein vitroandin vivogene expression profiles, we identified 351 and 255 genes as activated and repressed, respectively, during murine lung infection. As expected, numerous genes associated with virulent-phase growth were activated in the murine host, including pertussis toxin (PT), the PT secretion apparatus, and the type III secretion system. A significant number of genes encoding iron acquisition and heme uptake proteins were highly expressed during infection, supporting iron acquisition as critical forB. pertussissurvivalin vivo. Numerous metabolic genes were repressed during infection. Overall, these data shed light on the gene expression profile ofB. pertussisduring infection, and this method will facilitate efforts to understand how this pathogen causes infection.IMPORTANCEIn vitrogrowth conditions for bacteria do not fully recapitulate the host environment. RNA sequencing transcriptome analysis allows for the characterization of the infection gene expression profiles of pathogens in complex environments. Isolation of the pathogen from infected tissues is critical because of the large amounts of host RNA present in crude lysates of infected organs. A filtration method was developed that enabled enrichment of the pathogen RNA for RNA-seq analysis. The resulting data describe the “infection transcriptome” ofB. pertussisin the murine lung. This strategy can be utilized for pathogens in other hosts and, thus, expand our knowledge of what bacteria express during infection.

Hematopoietic stem and progenitor cell (HSPC) compartments are altered to direct immune responses to infection. Their roles during immunization are not well-described. To elucidate mechanisms for waning immunity following immunization with acellular vaccines (ACVs) against Bordetella pertussis (Bp), we tested the hypothesis that immunization with Bp ACVs and whole cell vaccines (WCVs) differ in directing the HSPC characteristics and immune cell development patterns that ultimately contribute to the types and quantities of cells produced to fight infection. Our data demonstrate that compared to control and ACV-immunized CD-1 mice, immunization with an efficacious WCV drives expansion of hematopoietic multipotent progenitor cells (MPPs), increases circulating white blood cells (WBCs), and alters the size and composition of lymphoid organs. In addition to MPPs, common lymphoid progenitor (CLP) proportions increase in the bone marrow of WCV-immunized mice, while B220+ cell proportions decrease. Upon subsequent infection, increases in maturing B cell populations are striking in WCV-immunized mice. RNAseq analyses of HSPCs revealed that WCV and ACV-immunized mice vastly differ in developing VDJ gene segment diversity. Moreover, gene set enrichment analyses demonstrate WCV-immunized mice exhibit unique gene signatures that suggest roles for interferon (IFN) induced gene expression. Also observed in naïve infection, these IFN stimulated gene (ISG) signatures point toward roles in cell survival, cell cycle, autophagy, and antigen processing and presentation. Taken together, these findings underscore the impact of vaccine antigen and adjuvant content on skewing and/or priming HSPC populations for immune response.

18Bordetella pertussis (B. pertussis) is the causative agent of pertussis (whooping cough). 19 Since the 1990s, pertussis has re-emerged in the United States despite an estimated 20 95% vaccine coverage. Our goal was to characterize neutrophil responses and gene 21 expression profiles of murine lungs in the context of vaccination and B. pertussis 22 challenge. We utilized a bioluminescent neutrophil mouse model (NECre luc) to track 23 neutrophil recruitment. NECre luc mice were immunized with whole cell vaccine (WCV), 24 acellular vaccine (ACV), or a truncated adenylate cyclase toxoid (RTX) vaccine. 25 Neutrophil recruitment was measured in live mice across time and corroborated by flow 26 cytometry and other data. WCV immunized mice showed signs of neutrophilia in response 27 to B. pertussis challenge. Mice immunized with either ACV or WCV cleared the challenge 28 infection; however immunization with RTX alone was not protective. RNA sequencing 29 revealed distinctive gene expression profiles for each immunization group. We observed 30 an increase in expression of genes associated with responses to infection, and changes 31 in expression of distinct genes in each vaccine group, providing a complex view of the 32 immune response to B. pertussis infection in mice. This study suggests that combination 33 of immunological analysis with transcriptomic profiling can facilitate discovery of pre-34 clinical correlates of protection for vaccine development.35 36 37 38 39 41Pertussis is a human disease primarily caused by a respiratory infection of the Gram-42 negative pathogen Bordetella pertussis (B. pertussis). The hallmark of pertussis is a distinctive 43 whooping cough. What is surprising about pertussis is that the cause of the cough has never been 44 elucidated, which highlights the fact that there are many under-researched aspects of this 45 disease. Aerosolized B. pertussis bacterium are inhaled and adhere to airway respiratory 46 epithelial cells through bacterial adhesins such as filamentous hemagglutinin (FHA), fimbriae and 47 pertactin 1 . After colonization B. pertussis express multiple toxins including pertussis toxin (PT) 48 and adenylate cyclase toxin (ACT). B. pertussis releases PT, which dysregulates the immune 49 response through ADP-ribosylation of the G-protein α-subunit of cytokine receptors present on a 50 range of leukocytes 2-5 . The secretion of PT has long range effects, ACT is thought to act locally 51 on host cells by converting ATP into supraphysiological levels of cAMP, further dysregulating the 52 host immune response 6 . 53In the 1940s, an effective whole cell vaccine (WCV) was developed and as a result, basic research 54 efforts on B. pertussis decreased. The WCVs were highly reactogenic and caused prolonged and 55 unusual crying after administration, hyporesponsivness, and febrile convulsions 7-9 . These issues 56 led to the development of acellular vaccines (ACV), known today as DTaP/Tdap (hereafter 57 referred to as ACV). The ACVs utilize an alum adjuvant and induce a Th2 response ...

No abstract

Pseudomonas aeruginosais an opportunistic pathogen that requires iron for growth and virulence, yet this nutrient is sequestered by the innate immune system during infection. When iron is limiting,P. aeruginosaexpresses the PrrF1 and PrrF2 small regulatory RNAs (sRNAs), which post-transcriptionally repress expression of non-essential iron-containing proteins thus sparing this nutrient for more critical processes. The genes for the PrrF1 and PrrF2 sRNAs are arranged in tandem on the chromosome, allowing for the transcription of a longer heme-responsive sRNA, termed PrrH. While the functions of PrrF1 and PrrF2 have been studied extensively, the role of PrrH inP. aeruginosaphysiology and virulence is not well understood. In this study, we performed transcriptomic and proteomic studies to identify the PrrH regulon. In shaking cultures, the pyochelin synthesis proteins were increased in two distinctprrHmutants compared to wild type, while the mRNAs for these proteins were not affected byprrHmutation. We identified complementarity between the PrrH sRNA and sequence upstream of thepchEmRNA, suggesting potential for PrrH to directly regulate expression of genes for pyochelin synthesis. We further showed thatpchEmRNA levels were increased in theprrHmutants when grown in static but not shaking conditions. Moreover, we discovered controlling for the presence of light was critical for examining the impact of PrrH onpchEexpression. As such, our study reports on the first likely target of the PrrH sRNA and highlights key environmental variables that will allow for future characterization of PrrH function.