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

Abstract

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.

Transcriptional profiling of human macrophages during infection withBordetella pertussis

“…The expression of bvgR regulator of virulence was reduced during the infection and, consequently, the expression of several virulence factors was also downregulated. This is in contrast with in vivo data obtained previously in mouse model of respiratory tract infection where expression of majority of virulence factors was increased during the infection of lungs [35,36,68]. Our observations indicate that opposed to B. pertussis cells isolated from mouse organs and facing systemic immune response, the intracellular B. pertussis cells switch from Bvg + to Bvg − phase during the intramacrophage phase.…”

“…Among those induced during infection were genes involved in iron transport and iron-sulphur cluster assembly indicating that intracellular B. pertussis cells face iron scarcity and oxidative stress. These results are in line with previous reports showing that iron acquisition and transport systems and iron-sulphur cluster sensors contribute to fitness of B. pertussis cells during in vivo infections [35,36,66,67]. Within the genes displaying reduced expression during infection were also those involved in pathogenesis.…”

“…Nevertheless, strongly modulated expression of dozens of cytokines and chemokines suggests that some of the observed effects result from cell-to-cell signalling within the population of infected macrophages. Importantly, this study indicates that in vitro expressions profiles of several genes and pathways in both human host cells and pathogen are in line with in vivo data obtained with mouse model of infection [34][35][36]66], although our data suggest higher importance of avirulent phase in intramacrophage infection than previously thought. Nevertheless, we are aware of certain limitations of our study and it is evident that we need to perform additional experiments to delineate the B. pertussisspecific activities modulating the host immune response and macrophage-specific responses induced by B. pertussis infection in order to clarify the importance of the intracellular phase for the infectious cycle of B. pertussis.…”

“…This study represents the first RNA-seq-based attempt to assess the global transcriptomic response of human phagocytes triggered by infection with B. pertussis cells. So far the transcriptional profiling of B. pertussis interactions with host cells was studied predominantly in mouse model and was focused either on the host [33,34] or the pathogen [35,36]. Nevertheless, mouse is not a natural host of B. pertussis and in our study we were interested in specific interaction with human macrophages rather than with complex mouse immune system.…”

Bordetella Type III Secretion Injectosome and Effector Proteins

“…The bsc -encoded genes of the Bordetella injectosome are induced during infection, and are responsive to blood or serum exposure, and increased CO 2 concentrations (Gaillard et al, 2011 ; Hester et al, 2012 ; Bibova et al, 2015 ; Gestal et al, 2018 ; Van Beek et al, 2018 ; Wong et al, 2019 ). Other stimuli that can activate T3SS expression and secretion in bordetellae are the stringent response induced by iron limitation and/or starvation for carbon source, as depicted in Figure 4 (Brickman et al, 2011 ; Kurushima et al, 2012b ; Hanawa et al, 2016 ).…”