Development of RNAseq methodologies to profile thein vivotranscriptome ofBordetella pertussisduring murine lung infection

Abstract

16Bordetella pertussis is an obligate human respiratory pathogen that causes the disease whooping 17 cough. A whole cell vaccine (DTP) was developed in the 1940s and was subsequently replaced 18 in the 1990s with a protein-based subunit acellular vaccine (DTaP; tdap). Today, we are 19 observing a resurgence of whooping cough due to evolution of the pathogen and waning vaccine 20 immunity. The use of vaccines decreased the need for basic research on this pathogen. As a 21 result, numerous questions on the basic pathogenesis of B. pertussis remain to be answered. 22Microarrays and more recently, RNA sequencing (RNAseq), have allowed the field to describe 23 the in vitro gene expression profiles of the pathogen growing in both virulent and avirulent phases; 24 however, no published studies have described an in vivo transcriptome of the pathogen. To 25 address this need, we have designed and evaluated workflows to characterize the in vivo 26 transcriptome of B. pertussis during infection of the murine lung. During our initial studies, we 27 observed that only 0.014% of the ~100 million 2x50bp illumina reads corresponded to the 28 pathogen, which is insufficient for analysis. Therefore, we developed a simple protocol to filter the 29 bacteria out of the tissue homogenates and separate bacterial cells from the host tissue. RNA is 30 then prepared, quantified, and the B. pertussis to host RNA ratio is determined. Here, we present 31 the protocol and discuss the uses and next directions for which this RNAseq workflow can be 32 applied. With this strategy we plan to fully characterize the B. pertussis transcriptome when the 33 pathogen is infecting the murine lung in order to identify expressed genes that encode potential 34 new vaccine antigens that will facilitate the development of the next generation of pertussis 35 vaccines.

Analysis of the In Vivo Transcriptome of Bordetella pertussis during Infection of Mice

“…Despite the limitations of that data set, we observed that the alcaligin siderophore biosynthesis genes were highly expressed by B. pertussis infecting the NSG mouse lung (30). Furthermore, we saw that most reads mapped to fhaB , which encodes FHA, suggesting that this gene could be highly expressed during infection.…”

“…Consequently, the NSG mouse also harbors defective macrophages and dendritic cells, with only neutrophils and monocytes unaffected by the mutations. With this in mind, we aimed to use the high bacterial burden in NSG mice to facilitate RNA-seq of infected murine lung (30). However, we were able to map only approximately 15,000 reads per 100 million to the B.…”

“…However, we were able to map only approximately 15,000 reads per 100 million to the B. pertussis genome (30). Despite the limitations of that data set, we observed that the alcaligin siderophore biosynthesis genes were highly expressed by B.…”

“…pertussis bacteria that passed through the filter. The filtration allowed for isolation of ∼50% of the viable bacteria from the infected lungs (data not shown) (30). To confirm that the filtration method allowed for detection of bacterial transcripts, we performed quantitative reverse transcription-PCR (qRT-PCR) analysis on B.…”

“…When we attempted to perform dual RNA-seq with B. pertussis, we identified an insufficient number of reads, and it was not possible to precisely characterize the bacterial pathogen’s in vivo transcriptome (30). In this study, we describe a simple filtration method that allowed us to isolate bacteria to facilitate characterization of the in vivo transcriptome of B.…”