Comparative genomics of Czech vaccine strains of Bordetella pertussis.

Bordetella pertussis is a strictly human pathogen causing the respiratory infectious disease called whooping cough or pertussis. B. pertussis adaptation to acellular pertussis vaccine pressure has been repeatedly highlighted, but recent data indicate that adaptation of circulating strains started already in the era of the whole cell pertussis vaccine (wP) use. We sequenced the genomes of five B. pertussis wP vaccine strains isolated in the former Czechoslovakia in the pre-wP (1954-1957) and early wP (1958-1965) eras, when only limited population travel into and out of the country was possible. Four isolates exhibit a similar genome organization and form a distinct phylogenetic cluster with a geographic signature. The fifth strain is rather distinct, both in genome organization and SNP-based phylogeny. Surprisingly, despite isolation of this strain before 1966, its closest sequenced relative appears to be a recent isolate from the US. On the genome content level, the five vaccine strains contained both new and already described regions of difference. One of the new regions contains duplicated genes potentially associated with transport across the membrane. The prevalence of this region in recent isolates indicates that its spread might be associated with selective advantage leading to increased strain fitness

Translational activation of rpoS mRNA by the non-coding RNA DsrA and Hfq does not require ribosome binding

At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, σS, involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind

Omics Analysis of Blood-Responsive Regulon in <i>Bordetella pertussis</i> Identifies a Novel Essential T3SS Substrate

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

A Bordetella pertussis MgtC homolog plays a role in the intracellular survival.

Bordetella pertussis, the causative agent of whooping cough, has the capability to survive inside the host cells. This process requires efficient adaptation of the pathogen to the intracellular environment and the associated stress. Among the proteins produced by the intracellular B. pertussis we identified a protein (BP0414) that shares homology with MgtC, a protein which was previously shown to be involved in the intracellular survival of other pathogens. To explore if BP0414 plays a role in B. pertussis intracellular survival a mutant strain defective in the production of this protein was constructed. Using standard in vitro growth conditions we found that BP0414 is required for B. pertussis growth under low magnesium availability or low pH, two environmental conditions that this pathogen might face within the host cell. Intracellular survival studies showed that MgtC is indeed involved in B. pertussis viability inside the macrophages. The use of bafilomycin A1, which inhibits phagosome acidification, abolished the survival defect of the mgtC deficient mutant strain suggesting that in intracellular B. pertussis the role of MgtC protein is mainly related to the bacterial adaptation to the acidic conditions found inside the of phagosomes. Overall, this work provides an insight into the importance of MgtC in B. pertussis pathogenesis and its contribution to bacterial survival within immune cells

A <i>Bordetella pertussis</i> MgtC homolog plays a role in the intracellular survival

Bordetella pertussis, the causative agent of whooping cough, has the capability to survive inside the host cells. This process requires efficient adaptation of the pathogen to the intracellular environment and the associated stress. Among the proteins produced by the intracellular B. pertussis we identified a protein (BP0414) that shares homology with MgtC, a protein which was previously shown to be involved in the intracellular survival of other pathogens. To explore if BP0414 plays a role in B. pertussis intracellular survival a mutant strain defective in the production of this protein was constructed. Using standard in vitro growth conditions we found that BP0414 is required for B. pertussis growth under low magnesium availability or low pH, two environmental conditions that this pathogen might face within the host cell. Intracellular survival studies showed that MgtC is indeed involved in B. pertussis viability inside the macrophages. The use of bafilomycin A1, which inhibits phagosome acidification, abolished the survival defect of the mgtC deficient mutant strain suggesting that in intracellular B. pertussis the role of MgtC protein is mainly related to the bacterial adaptation to the acidic conditions found inside the of phagosomes. Overall, this work provides an insight into the importance of MgtC in B. pertussis pathogenesis and its contribution to bacterial survival within immune cells.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones Industriale

Hfq modulates global protein pattern and stress response in Bordetella pertussis

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.Fil: Alvarez Hayes, Jimena. 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: Surmann, Kristin. University Medicine Greifswald; AlemaniaFil: Lamberti, Yanina Andrea. 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: Depke, Maren. University Medicine Greifswald; AlemaniaFil: Dhople, Vishnu. University Medicine Greifswald; AlemaniaFil: Blancá, Bruno Martin. 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: Ruiz, María Esperanza. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Vecerek, Branislav. Institute of Microbiology of the ASCR; República ChecaFil: Schmidt, Frank. University Medicine Greifswald; AlemaniaFil: Völker, Uwe. University Medicine Greifswald; AlemaniaFil: 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