Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA.In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Bordetella pertussis * metabolismus MeSH
- chromatografie kapalinová MeSH
- lidé MeSH
- pertuse * MeSH
- regulace genové exprese u bakterií MeSH
- sigma faktor genetika MeSH
- tandemová hmotnostní spektrometrie MeSH
- umělá inteligence MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Apart from oxygenic photosynthesis, the extent of manganese utilization in bacteria varies from species to species and also appears to depend on external conditions. This observation is in striking contrast to iron, which is similar to manganese but essential for the vast majority of bacteria. To adequately explain the role of manganese in pathogens, we first present in this review that the accumulation of molecular oxygen in the Earth's atmosphere was a key event that linked manganese utilization to iron utilization and put pressure on the use of manganese in general. We devote a large part of our contribution to explanation of how molecular oxygen interferes with iron so that it enhances oxidative stress in cells, and how bacteria have learned to control the concentration of free iron in the cytosol. The functioning of iron in the presence of molecular oxygen serves as a springboard for a fundamental understanding of why manganese is so valued by bacterial pathogens. The bulk of this review addresses how manganese can replace iron in enzymes. Redox-active enzymes must cope with the higher redox potential of manganese compared to iron. Therefore, specific manganese-dependent isoenzymes have evolved that either lower the redox potential of the bound metal or use a stronger oxidant. In contrast, redox-inactive enzymes can exchange the metal directly within the individual active site, so no isoenzymes are required. It appears that in the physiological context, only redox-inactive mononuclear or dinuclear enzymes are capable of replacing iron with manganese within the same active site. In both cases, cytosolic conditions play an important role in the selection of the metal used. In conclusion, we summarize both well-characterized and less-studied mechanisms of the tug-of-war for manganese between host and pathogen.
The ability of bacterial pathogens to acquire essential micronutrients is critical for their survival in the host environment. Manganese plays a complex role in the virulence of a variety of pathogens due to its function as an antioxidant and enzymatic cofactor. Therefore, host cells deprive pathogens of manganese to prevent or attenuate infection. Here, we show that evolution of the human-restricted pathogen Bordetella pertussis has selected for an inhibitory duplication within a manganese exporter of the calcium:cation antiporter superfamily. Intriguingly, upon exposure to toxic levels of manganese, the nonfunctional exporter becomes operative in resister cells due to a unique reverse adaptation mechanism. However, compared with wild-type (wt) cells, the resisters carrying a functional copy of the exporter displayed strongly reduced intracellular levels of manganese and impaired growth under oxidative stress. Apparently, inactivation of the manganese exporter and the resulting accumulation of manganese in the cytosol benefited the pathogen by improving its survival under stress conditions. The inhibitory duplication within the exporter gene is highly conserved among B. pertussis strains, absent from all other Bordetella species and from a vast majority of organisms across all kingdoms of life. Therefore, we conclude that inactivation of the exporter gene represents an exceptional example of a flexible genome decay strategy employed by a human pathogen to adapt to its exclusive host. IMPORTANCE Bordetella pertussis, a respiratory pathogen restricted to humans, continuously adapts its genome to its exclusive host. We show that speciation of this reemerging pathogen was accompanied by loss of function of the manganese exporter. Intriguingly, the functionality of the exporter can be restored in the presence of toxic levels of manganese by a unique genetic modification. However, compared with the wt strain, the strain carrying the functional exporter failed to resist the oxidative stress in vitro. Thus, our data demonstrate that inactivation of the exporter resulting in manganese accumulation assists B. pertussis in adaptation to oxidative stress. We conclude that this sophisticated process of reverse adaptation enables B. pertussis to adjust to rapidly changing environments by facilitating its resistance to both manganese toxicity and manganese scarcity.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Bordetella pertussis účinky léků genetika patogenita MeSH
- faktory virulence genetika MeSH
- lidé MeSH
- mangan toxicita MeSH
- oxidační stres MeSH
- pertuse prevence a kontrola MeSH
- regulace genové exprese u bakterií účinky léků MeSH
- virulence účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Despite high vaccination coverage, pertussis is increasing in many industrialized countries, including the Czech Republic. To better understand Bordetella pertussis resurgence, we analyzed historic strains and recent clinical isolates by using a comparative omics approach. Whole-genome sequencing showed that historic and recent isolates of B. pertussis have substantial variation in genome organization and form separate phylogenetic clusters. Subsequent RNA sequence analysis and liquid chromatography with mass tandem spectrometry analyses showed that these variations translated into discretely separated transcriptomic and proteomic profiles. When compared with historic strains, recent isolates showed increased expression of flagellar genes and genes involved in lipopolysaccharide biosynthesis and decreased expression of polysaccharide capsule genes. Compared with reference strain Tohama I, all strains had increased expression and production of the type III secretion system apparatus. We detected the potential link between observed effects and insertion sequence element-induced changes in gene context only for a few genes.
- MeSH
- Bordetella pertussis * genetika MeSH
- fylogeneze MeSH
- lidé MeSH
- pertuse * epidemiologie MeSH
- pertusová vakcína MeSH
- proteomika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Bacterial pathogens sense specific cues associated with different host niches and integrate these signals to appropriately adjust the global gene expression. Bordetella pertussis is a Gram-negative, strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Though B. pertussis does not cause invasive infections, previous results indicated that this reemerging pathogen responds to blood exposure. Here, omics RNA-seq and LC-MS/MS techniques were applied to determine the blood-responsive regulon of B. pertussis. These analyses revealed that direct contact with blood rewired global gene expression profiles in B. pertussis as the expression of almost 20% of all genes was significantly modulated. However, upon loss of contact with blood, the majority of blood-specific effects vanished, with the exception of several genes encoding the T3SS-secreted substrates. For the first time, the T3SS regulator BtrA was identified in culture supernatants of B. pertussis. Furthermore, proteomic analysis identified BP2259 protein as a novel secreted T3SS substrate, which is required for T3SS functionality. Collectively, presented data indicate that contact with blood represents an important cue for B. pertussis cells.
- MeSH
- anotace sekvence MeSH
- bakteriální proteiny metabolismus MeSH
- Bordetella pertussis fyziologie MeSH
- chromatografie kapalinová MeSH
- faktory virulence MeSH
- genomika * metody MeSH
- lidé MeSH
- proteomika * metody MeSH
- regulace genové exprese u bakterií MeSH
- sekreční systém typu III genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- tandemová hmotnostní spektrometrie MeSH
- transkriptom MeSH
- virulence MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The BvgS/BvgA two-component system controls expression of ∼550 genes of Bordetella pertussis, of which, ∼245 virulence-related genes are positively regulated by the BvgS-phosphorylated transcriptional regulator protein BvgA (BvgA∼P). We found that a single G-to-T nucleotide transversion in the 5'-untranslated region (5'-UTR) of the rplN gene enhanced transcription of the ribosomal protein operon and of the rpoA gene and provoked global dysregulation of B. pertussis genome expression. This comprised overproduction of the alpha subunit (RpoA) of the DNA-dependent RNA polymerase, downregulated BvgA and BvgS protein production, and impaired production and secretion of virulence factors by the mutant. Nonetheless, the mutant survived like the parental bacteria for >2 weeks inside infected primary human macrophages and persisted within infected mouse lungs for a longer period than wild-type B. pertussis These observations suggest that downregulation of virulence factor production by bacteria internalized into host cells may enable persistence of the whooping cough agent in the airways.IMPORTANCE We show that a spontaneous mutation that upregulates transcription of an operon encoding ribosomal proteins and causes overproduction of the downstream-encoded α subunit (RpoA) of RNA polymerase causes global effects on gene expression levels and proteome composition of Bordetella pertussis Nevertheless, the resulting important downregulation of the BvgAS-controlled expression of virulence factors of the whooping cough agent did not compromise its capacity to persist for prolonged periods inside primary human macrophage cells, and it even enhanced its capacity to persist in infected mouse lungs. These observations suggest that the modulation of BvgAS-controlled expression of virulence factors may occur also during natural infections of human airways by Bordetella pertussis and may possibly account for long-term persistence of the pathogen within infected cells of the airways.
- Publikační typ
- časopisecké články MeSH
B. pertussis is the etiological agent of whooping cough, a highly contagious respiratory disease which remains uncontrolled worldwide. Understanding how this pathogen responds to the environmental changes and adapts to different niches found inside the host might contribute to gain insight into bacterial pathogenesis. Comparative analyses of previous transcriptomic and proteomic data suggested that post-transcriptional regulatory mechanisms modulate B. pertussis virulence in response to iron availability. Iron scarcity represents one of the major stresses faced by bacterial pathogens inside the host. In this study, we used gel-free nanoLC-MS/MS-based proteomics to investigate whether Hfq, a highly conserved post-transcriptional regulatory protein, is involved in B. pertussis adaptation to low iron environment. To this end, we compared the protein profiles of wild type B. pertussis and its isogenic hfq deletion mutant strain under iron-replete and iron-depleted conditions. Almost of 33% of the proteins identified under iron starvation was found to be Hfq-dependent. Among them, proteins involved in oxidative stress tolerance and virulence factors that play a key role in the early steps of host colonization and bacterial persistence inside the host cells. Altogether these results suggest that Hfq shapes the infective phenotype of B. pertussis. SIGNIFICANCE: In the last years, it became evident that post-transcriptional regulation of gene expression in ba cteria plays a central role in host-pathogen interactions. Hfq is a bacterial protein that regulates gene expression at post-transcriptional level found pivotal in the establishment of successful infections. In this study, we investigated the role of Hfq in Bordetella pertussis response to iron starvation, one of the main stresses imposed by the host. The data demonstrate that Hfq regulates the abundance of a significant number of B. pertussis proteins in response to iron starvation. Among them, virulence factors and proteins involved in oxidative stress tolerance, key players in host colonization and intracellular bacterial survival. Altogether, our results suggest a relevant role of Hfq in B. pertussis adaptation to the different niches found inside the host eventually granting bacterial pathogenesis.
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Bordetella pertussis is the causative agent of human whooping cough (pertussis), a highly contagious respiratory disease that remains to be the least controlled vaccine-preventable infectious disease. Despite generalized vaccination, pertussis is again on the rise in highly vaccinated populations including Czech Republic. The increase in pertussis cases has been mostly attributed to short-lived immunity induced by the acellular vaccine and escape from immunity due to pathogen adaptation and antigen variation. Therefore, using comparative genomics we will analyze Czech clinical isolates of B. pertussis collected during the recent period 2008 - 2015. Importantly, genomic sequences, expression and production of virulence factors will be compared with those of B. pertussis vaccine strains. In addition, using proteomics and immunoprecipitation we will apply recent human sera of infected patients for identification of novel immunogenic antigens of B. pertussis.
Bordetella pertussis je lidský patogen, který je původcem černého kašle, vysoce nakažlivé respirační choroby, jejíž výskyt je v současné době na vzestupu a to i v ekonomicky vyspělých zemích s vysokou proočkovaností jako je Česká republika. Tento vzestup je přičítán zejména přechodu z celobuněčných vakcín na vakcíny acelulární. Acelulární vakcíny jsou sice ve srovnání s celobuněčnými méně reaktogenní, nicméně poskytují sníženou a kratší ochranu před nákazou. Navíc došlo u cirkulujících kmenů B. pertussis v reakci na antigeny obsažené ve vakcínách ke genotypovým změnám. Naším cílem je analyzovat celogenomové sekvence a produkci faktorů virulence v klinických izolátech získaných na území České republiky v letech 2008 – 2015. Pro srovnávací analýzu budou využity i původní vakcinační kmeny používané k výrobě celobuněčné vakcíny. Dále pomocí proteomiky a imunoprecipitace použijeme séra pacientů nakažených černým kašlem k identifikaci nových imunogenních antigenů produkovaných současnými kmeny B. pertussis.
- MeSH
- Bordetella pertussis genetika patogenita MeSH
- faktory virulence rodu Bordetella analýza MeSH
- lidé MeSH
- pertuse genetika prevence a kontrola MeSH
- sekvenování celého genomu metody MeSH
- virulence MeSH
- Check Tag
- lidé MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- genetika, lékařská genetika
- pneumologie a ftizeologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Bordetella pertussis, a strictly human re-emerging pathogen and the causative agent of whooping cough, exploits a broad variety of virulence factors to establish efficient infection. Here, we used RNA sequencing to analyse the changes in gene expression profiles of human THP-1 macrophages resulting from B. pertussis infection. In parallel, we attempted to determine the changes in intracellular B. pertussis-specific transcriptomic profiles resulting from interaction with macrophages. Our analysis revealed that global gene expression profiles in THP-1 macrophages are extensively rewired 6 h post-infection. Among the highly expressed genes, we identified those encoding cytokines, chemokines, and transcription regulators involved in the induction of the M1 and M2 macrophage polarization programmes. Notably, several host genes involved in the control of apoptosis and inflammation which are known to be hijacked by intracellular bacterial pathogens were overexpressed upon infection. Furthermore, in silico analyses identified large temporal changes in expression of specific gene subsets involved in signalling and metabolic pathways. Despite limited numbers of the bacterial reads, we observed reduced expression of majority of virulence factors and upregulation of several transcriptional regulators during infection suggesting that intracellular B. pertussis cells switch from virulent to avirulent phase and actively adapt to intracellular environment, respectively.
- MeSH
- Bordetella pertussis fyziologie MeSH
- buněčné linie MeSH
- genová ontologie MeSH
- genové regulační sítě MeSH
- interakce hostitele a patogenu genetika imunologie MeSH
- kultivované buňky MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- makrofágy imunologie metabolismus mikrobiologie MeSH
- pertuse genetika imunologie virologie MeSH
- regulace genové exprese MeSH
- reprodukovatelnost výsledků MeSH
- stanovení celkové genové exprese * metody MeSH
- transkriptom * MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
