Loic Coutte

Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway.

Proteins of Gram‐negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place. The synthesis of a precursor may be a strategy to maintain secretion competence while preventing aggregation or premature folding (especially for large proteins). The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its ∼130 kDa C‐terminal intramolecular chaperone domain. We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA. SphB1 is a large exported protein with a subtilisin‐like domain and a C‐terminal domain typical of bacterial autotransporters. SphB1 is the first described subtilisin‐like protein that serves as a specialized maturation protease in a secretion pathway of Gram‐negative bacteria. This is reminiscent of pro‐protein convertases of eukaryotic cells.

Role of Adhesin Release for Mucosal Colonization by a Bacterial Pathogen

Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors called adhesins. To exert these functions, adhesins are typically surface-exposed, although, surprisingly, some are also released into the extracellular milieu, the relevance of which has previously not been studied. To address the role of adhesin release in pathogenesis, we used Bordetella pertussis as a model, since its major adhesin, filamentous hemagglutinin (FHA), partitions between the bacterial surface and the extracellular milieu. FHA release depends on its maturation by the specific B. pertussis protease SphB1. We constructed SphB1-deficient mutants and found that they were strongly affected in their ability to colonize the mouse respiratory tract, although they adhered even better to host cells in vitro than their wild-type parent strain. The defect in colonization could be overcome by prior nasal instillation of purified FHA or by coinfection with FHA-releasing B. pertussis strains, but not with SphB1-producing FHA-deficient strains, ruling out a nonspecific effect of SphB1. These results indicate that the release of FHA is important for colonization, as it may facilitate the dispersal of bacteria from microcolonies and the binding to new sites in the respiratory tract.

New Virulence-Activated and Virulence-Repressed Genes Identified by Systematic Gene Inactivation and Generation of Transcriptional Fusions in Bordetella pertussis

An in silico scan of the partially completed genome sequence of Bordetella pertussis and analyses of transcriptional fusions generated with a new integrational vector were used to identify new potential virulence genes. The genes encoding a putative siderophore receptor, adhesins, and an autotransporter protein appeared to be regulated in a manner similar to Bordetella virulence genes by the global virulence regulator BvgAS. In contrast, the gene encoding a putative intimin-like protein appeared to be repressed under conditions of virulence.

The ins and outs of pertussis toxin

Pertussis toxin, produced and secreted by the whooping cough agent Bordetella pertussis, is one of the most complex soluble bacterial proteins. It is actively secreted through the B. pertussis cell envelope by the Ptl secretion system, a member of the widespread type IV secretion systems. The toxin is composed of five subunits (named S1 to S5 according to their decreasing molecular weights) arranged in an A–B structure. The A protomer is composed of the enzymatically active S1 subunit, which catalyzes ADP‐ribosylation of the α subunit of trimeric G proteins, thereby disturbing the metabolic functions of the target cells, leading to a variety of biological activities. The B oligomer is composed of 1S2:1S3:2S4:1S5 and is responsible for binding of the toxin to the target cell receptors and for intracellular trafficking via receptor‐mediated endocytosis and retrograde transport. The toxin is one of the most important virulence factors of B. pertussis and is a component of all current vaccines against whooping cough.

Surface anchoring of bacterial subtilisin important for maturation function

Many extracytoplasmic proteins undergo proteolytic processing during secretion, which is essential to their maturation. These post‐translational modifications are carried out by specific enzymes whose subcellular localization is important for function. We have described a maturation subtilisin in Gram‐negative Bordetella pertussis, the autotransporter SphB1. SphB1 catalyses the maturation of the precursor of the adhesin filamentous haemagglutinin (FHA) at the bacterial surface, in addition to the processing of its own precursor. Here, we show that the outer membrane anchor of SphB1 is crucial to its function, as evidenced by the lack of FHA maturation in a strain releasing a variant of SphB1 into the milieu. In contrast, surface association is not required for automaturation of SphB1. The surface retention of mature SphB1 is mediated by lipidation of the protein. The tethered protease appears to be stabilized by unusual Gly‐ and Pro‐rich motifs at the N‐terminus of the protein. This represents a new mode of localization for a protease involved in protein secretion.

The multifaceted RisA regulon of Bordetella pertussis.

The whooping cough agent Bordetella pertussis regulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of the bvgR gene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that the expression of almost all vrgs depends on RisA, another transcriptional regulator. We also show that some vags are surprisingly no longer modulated by MgSO4 in the risA− background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD60N and the phosphomimetic RisAD60E analogues. Thus the RisA regulon adds a new layer of complexity to B. pertussis virulence gene regulation.

Primary transcriptome analysis reveals importance of IS elements for the shaping of the transcriptional landscape of Bordetella pertussis

ABSTRACT 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.

Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis

The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen.

Construction and evaluation of Bordetella pertussis live attenuated vaccine strain BPZE1 producing Fim3.

Pertussis or whooping cough is currently the most prevalent vaccine-preventable childhood disease despite >85% global vaccination coverage. In recent years incidence has greatly increased in several high-income countries that have switched from the first-generation, whole-cell vaccine to the newer acellular vaccines, calling for improved vaccination strategies with better vaccines. We have developed a live attenuated pertussis vaccine candidate, called BPZE1, which is currently in clinical development. Unlike other pertussis vaccines, BPZE1 has been shown to provide strong protection against infection by the causative agent of pertussis, Bordetella pertussis, in non-human primates. BPZE1 is a derivative of the B. pertussis strain Tohama I, which produces serotype 2 (Fim2) but not serotype 3 fimbriae (Fim3). As immune responses to fimbriae are likely to contribute to protection, we constructed a BPZE1 derivative, called BPZE1f3, that produces both serotypes of fimbriae. Whereas nasal vaccination of mice with BPZE1 induced antibodies to Fim2 but not to Fim3, vaccination with BPZE1f3 elicited antibodies to both Fim2 and Fim3 at approximately the same level. In mice, both BPZE1 and BPZE1f3 provided equal levels of protection against clinical isolates that either produce Fim2 alone, both Fim2 and Fim3, or no fimbriae. However, vaccination with BPZE1f3 provided significantly stronger protection against Fim3-only producing B. pertussis than vaccination with BPZE1, indicating that immune responses to fimbriae contribute to serotype-specific protection against B. pertussis infection.

Distinct virulence ranges for infection of mice by Bordetella pertussis revealed by engineering of the sensor-kinase BvgS

The whooping cough agent Bordetella pertussis coordinately regulates the expression of its virulence factors with the two-component system BvgAS. In laboratory conditions, specific chemical modulators are used to trigger phenotypic modulation of B. pertussis from its default virulent Bvg+ phase to avirulent Bvg- or intermediate Bvgi phases, in which no virulence factors or only a subset of them are produced, respectively. Whether phenotypic modulation occurs in the host remains unknown. In this work, recombinant B. pertussis strains harboring BvgS variants were tested in a mouse model of infection and analyzed using transcriptomic approaches. Recombinant BP-BvgΔ65, which is in the Bvgi phase by default and can be up-modulated to the Bvg+ phase in vitro, could colonize the mouse nose but was rapidly cleared from the lungs, while Bvg+-phase strains colonized both organs for up to four weeks. These results indicated that phenotypic modulation, which might have restored the full virulence capability of BP-BvgΔ65, does not occur in mice or is temporally or spatially restricted and has no effect in those conditions. Transcriptomic analyses of this and other recombinant Bvgi and Bvg+-phase strains revealed that two distinct ranges of virulence gene expression allow colonization of the mouse nose and lungs, respectively. We also showed that a recombinant strain expressing moderately lower levels of the virulence genes than its wild type parent was as efficient at colonizing both organs. Altogether, genetic modifications of BvgS generate a range of phenotypic phases, which are useful tools to decipher host-pathogen interactions.