Bordetella pertussis is the causative agent of whooping cough, a respiratory disease still considered as a major public health threat and for which recent re-emergence has been observed. Constant reshuffling of Bordetella pertussis genome organization was observed during evolution. These rearrangements are essentially mediated by Insertion Sequences (IS), a mobile genetic elements present in more than 230 copies in the genome, which are supposed to be one of the driving forces enabling the pathogen to escape from vaccine-induced immunity.Here we use high-throughput sequencing approaches (RNA-seq and differential RNA-seq), to decipher Bordetella pertussis transcriptome characteristics and to evaluate the impact of IS elements on transcriptome architecture. Transcriptional organization was determined by identification of transcription start sites and revealed also a large variety of non-coding RNAs including sRNAs, leaderless mRNAs or long 3′ and 5′UTR including seven riboswitches. Unusual topological organizations, such as overlapping 5′- or 3′-extremities between oppositely orientated mRNA were also unveiled. The pivotal role of IS elements in the transcriptome architecture and their effect on the transcription of neighboring genes was examined. This effect is mediated by the introduction of IS harbored promoters or by emergence of hybrid promoters. This study revealed that in addition to their impact on genome rearrangements, most of the IS also impact on the expression of their flanking genes. Furthermore, the transcripts produced by IS are strain-specific due to the strain to strain variation in IS copy number and genomic context.

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Bordetella pertussis, the causative agent of human whooping cough (pertussis) produces a complex array of virulence factors in order to establish efficient infection in the host. The RNA chaperone Hfq and small regulatory RNAs are key players in posttranscriptional regulation in bacteria and have been shown to play an essential role in virulence of a broad spectrum of bacterial pathogens. This study represents the first attempt to characterize the Hfq regulon of the human pathogen B. pertussis under laboratory conditions as well as upon passage in the host and indicates that loss of Hfq has a profound effect on gene expression in B. pertussis. Comparative transcriptional profiling revealed that Hfq is required for expression of several virulence factors in B. pertussis cells including the Type III secretion system (T3SS). In striking contrast to the wt strain, T3SS did not become operational in the hfq mutant passaged either through mice or macrophages thereby proving that Hfq is required for the functionality of the B. pertussis T3SS. Likewise, expression of virulence factors vag8 and tcfA encoding autotransporter and tracheal colonization factor, respectively, was strongly reduced in the hfq mutant. Importantly, for the first time we demonstrate that B. pertussis T3SS can be activated upon contact with macrophage cells in vitro.

Bordetella pertussis, the etiological agent of whooping cough, belongs to the bacterial pathogens first described in the so-called golden era of microbiology more than 100 years ago. In the course of the following decades, several other closely related pathogens were described which are nowadays classified in the genus Bordetella together with B. pertussis. These are the human and animal pathogens B. parapertussis, B. bronchiseptica and B. avium which are of high medical or veterinary interest, and which, together with B. pertussis, are referred to as the "classical" Bordetella species. Only in the past 15 years, several additional species were classified in the genus, frequently isolated from patients with underlying disease, animals or from the environment. Very little is known about most of these bacteria. In the present review, the current knowledge about these "new" Bordetella species is briefly summarized.

is the causative agent of human whooping cough, a highly contagious respiratory disease which despite vaccination programs remains the major cause of infant morbidity and mortality. The requirement of the RNA chaperone Hfq for virulence of suggested that Hfq-dependent small regulatory RNAs are involved in the modulation of gene expression. High-throughput RNA sequencing revealed hundreds of putative noncoding RNAs including the RgtA sRNA. Abundance of RgtA is strongly decreased in the absence of the Hfq protein and its expression is modulated by the activities of the two-component regulatory system BvgAS and another response regulator RisA. Whereas RgtA levels were elevated under modulatory conditions or in the absence of genes, deletion of the gene completely abolished RgtA expression. Profiling of the Δ mutant in the genetic background identified the gene encoding a periplasmic amino acid-binding protein of an ABC transporter as a possible target gene. The results of site-directed mutagenesis and in silico analysis indicate that RgtA base-pairs with the region upstream of the start codon of the mRNA and thereby weakens the BP3831 protein production. Furthermore, our data suggest that the function of the BP3831 protein is related to transport of glutamate, an important metabolite in the physiology. We propose that the BvgAS/RisA interplay regulates the expression of RgtA which upon infection, when glutamate might be scarce, attenuates translation of the glutamate transporter and thereby assists in adaptation of the pathogen to other sources of energy.

Haemophilus influenzae is a Gram-negative bacillus and a frequent commensal of the human nasopharynx. Earlier work demonstrated that in H. influenzae type b, l-lactate metabolism is associated with serum resistance and in vivo survival of the organism. To further gain insight into lactate utilization of the non-typeable (NTHi) isolate 2019 and laboratory prototype strain Rd KW20, deletion mutants of the l-lactate dehydrogenase (lctD) and permease (lctP) were generated and characterized. It is shown, that the apparent KM of l-lactate uptake is 20.1 μM as determined for strain Rd KW20. Comparison of the COPD isolate NTHi 2019-R with the corresponding lctP knockout strain for survival in human serum revealed no lactate dependent serum resistance. In contrast, we observed a 4-fold attenuation of the mutant strain in a murine model of nasopharyngeal colonization. Characterization of lctP transcriptional control shows that the lactate utilization system in H. influenzae is not an inductor inducible system. Rather negative feedback regulation was observed in the presence of l-lactate and this is dependent on the ArcAB regulatory system. Additionally, for 2019 it was found that lactate may have signaling function leading to increased cell growth in late log phase under conditions where no l-lactate is metabolized. This effect seems to be ArcA independent and was not observed in strain Rd KW20. We conclude that l-lactate is an important carbon-source and may act as host specific signal substrate which fine tunes the globally acting ArcAB regulon and may additionally affect a yet unknown signaling system and thus may contribute to enhanced in vivo survival.

IMPORTANCEIn the United States, Black and Hispanic children have higher rates of asthma and asthma-related morbidity compared with White children and disproportionately reside in communities with economic deprivation.OBJECTIVE To determine the extent to which neighborhood-level socioeconomic indicators explain racial and ethnic disparities in childhood wheezing and asthma. DESIGN, SETTING, AND PARTICIPANTSThe study population comprised children in birth cohorts located throughout the United States that are part of the Children's Respiratory and Environmental Workgroup consortium. Cox proportional hazard models were used to estimate hazard ratios (HRs) of asthma incidence, and logistic regression was used to estimate odds ratios of early and persistent wheeze prevalence accounting for mother's education, parental asthma, smoking during pregnancy, child's race and ethnicity, sex, and region and decade of birth.EXPOSURES Neighborhood-level socioeconomic indicators defined by US census tracts calculated as z scores for multiple tract-level variables relative to the US average linked to participants' birth record address and decade of birth. The parent or caregiver reported the child's race and ethnicity.MAIN OUTCOMES AND MEASURES Prevalence of early and persistent childhood wheeze and asthma incidence. RESULTSOf 5809 children, 46% reported wheezing before age 2 years, and 26% reported persistent wheeze through age 11 years. Asthma prevalence by age 11 years varied by cohort, with an overall median prevalence of 25%. Black children (HR, 1.47; 95% CI, and Hispanic children (HR, 1.29; 95% CI, 1.09-1.53) were at significantly increased risk for asthma incidence compared with White children, with onset occurring earlier in childhood. Children born in tracts with a greater proportion of low-income households, population density, and poverty had increased asthma incidence. Results for early and persistent wheeze were similar. In effect modification analysis, census variables did not significantly modify the association between race and ethnicity and risk for asthma incidence; Black and Hispanic children remained at higher risk for asthma compared with White children across census tracts socioeconomic levels. CONCLUSIONS AND RELEVANCEAdjusting for individual-level characteristics, we observed neighborhood socioeconomic disparities in childhood wheeze and asthma. Black and Hispanic children had more asthma in neighborhoods of all income levels. Neighborhood-and individual-level characteristics and their root causes should be considered as sources of respiratory health inequities.