Structure activity characterization of Bordetella petrii lipid A, from environment to human isolates

Highlights

• First structural characterization of Bordetella petrii lipid A.

• Comparison of lipopolysaccharides isolated from the environmental strain, with those of human isolates.

• B. petrii lipid A structures related to their cytokine induction capacities.

Abstract

Bordetella petrii, a facultative anaerobic species, is the only known member of the Bordetella genus with environmental origin. However it was also recently isolated from humans. The structures of the B. petrii lipid A moieties of the endotoxins were characterized here for the first time for an environmental strain and compared to that of human isolates. Characterization was achieved using chemical analyses, gas chromatography–mass spectrometry, and Matrix Assisted Laser Desorption Ionisation mass spectrometry. The analyses revealed that the different lipid A structures contain a common bisphosphorylated β-(1→6)-linked d-glucosamine disaccharide with hydroxytetradecanoic acid in amide as well at the C-3′ in ester linkages. Similar to Bordetella pertussis and Bordetella bronchiseptica lipids A, the hydroxytetradecanoic acid at the C-2′ position was substituted by tetradecanoic acid. Unlike B. pertussis, the hydroxytetradecanoic acid at the C-2 position was substituted with either 12:0 or 14:0 and/or their 2-OH forms. Depending on the environmental or human origin the structures differed in the length and degree of fatty acid acylation and impacted the IL-6 and TNF-α inflammatory responses tested. In one isolate we showed the presence at the C-3 position of the short-chain 10:0(3-OH), which according to our previous analyses is more characteristic of the human pathogens in the genus like B. pertussis and Bordetella parapertussis.

Introduction

The Bordetella genus consists of nine species, which includes several well-known pathogens capable to colonizing the upper respiratory tracts of mammals. The so-called “classical” species consist of Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, whereas the more “recent” ones are Bordetella avium, Bordetella hinzii, Bordetella holmesii, Bordetella trematum, Bordetella ansorpii and Bordetella petrii [1], [2]. Among these the most extensively studied include the first three. Although these three species are genetically very closely related [3], their LPS molecules display significant differences [4]. B. pertussis has long been recognised as the pathogen that infects only humans and is the causative agent of whooping cough in infants and adults [5]. Some B. parapertussis isolates are adapted to the human host and cause whooping cough, while others are adapted to the ovine host causing chronic pneumonia [6]. B. bronchiseptica, on the other hand colonizes the respiratory tract of a large number of animal hosts and can be symptomatic (Kennel cough, atrophic rhinitis, bronchopneumonia etc.) or asymptomatic and chronic. Occasionally, human infections with B. bronchiseptica have been reported for infants and patients with underlying disease or persons in close contact with animals. The agents of whooping cough - B. pertussis and a specific lineage of B. parapertussis – are assumed to have evolved independently from a B. bronchiseptica-like ancestor [7]. Along with the adaptation to human beings as the only host, genome reduction, accompanied by proliferation of Insertion Sequence (IS) elements, has played a fundamental role in the evolution of the pathogenic species [3], [8]. The genome sequence of B. avium 197 N has been determined and confirmed its quite distant relationship with the species of the B. bronchiseptica cluster [9].

B. petrii is the only known facultative anaerobic species of the genus with an environmental origin [10]. Interestingly, it was recently isolated from humans having different infections, including respiratory ones [11], [12], [13]. Even though other Bordetella species share many aspects of pathogenicity, they have distinct host ranges and cause different pathologies, which could be related to their surface components [14]. B. petrii was assigned to the Bordetella genus based on comparative 16S rDNA sequence analysis, DNA base composition, DNA–DNA hybridization experiments, and several metabolic properties [10].

Sequencing and annotation of B. petrii DSM12804 genome helped to understand this pathogen virulence mechanisms at the molecular level [15]. B. petrii is endowed with several virulence factors for plant and animal hosts such as adhesins (e.g. Fha, fimbriae) and a two-component complex regulator system, however, it does not contain any of the genes encoding pertussis toxin [15]. B petrii genome is also characterised by the presence of large mobile genomic islands encoding accessory metabolic functions [16]. However, few data are available for the LPS structure of B. petrii. Zelazny et al. [17] characterised B. petrii clinical isolates associated with different pathologies and observed differences between isolates (growth, antibiotic susceptibility and recognition by patient's antibodies) and for one isolate defect in the lipopolysaccharide O-antigen.

Lipopolysaccharides are the major outer-membrane components of many Gram-negative bacteria, and when toxic, act as pathogen-associated molecular pattern (PAMPs). LPS structures are very heterogeneous and multi-charged molecules from about 2 to 20 kDa.

They usually consists of a highly variable O-specific antigen characteristic of the species, a less variable core oligosaccharide of about 10 sugars, more similar from one species to another in a genus, and a relatively conserved lipidic moiety called lipid A. Some LPSs are displaying an outer core made of one or more oligosaccharide branches and no O-chain, they are referred to as lipooligosaccharides (LOS). Some examples are the LOS of B. pertussis [18], Neisseria meningitidis [14], and Haemophilus influenzae [14].

LPS, or LOS, form a ligand with the TLR4-MD2-CD14 complex, present on many cell types [19], [20]. This results to the release of cytokines, thereby initiating inflammatory and immune host responses [21]. Most of the biological activities of LPS have been associated with their lipid A that can be beneficial at low doses, for example stimulating the host immune system, or deleterious at higher doses, often leading to endotoxic shock and death but the polysaccharide fraction is also important for presentation of this structure to MD2 and TLR-4. Lipid A structures, elaborated during the biosynthesis pathway, can be modified after being exported to the external membrane via post-translational mechanisms such as acylation, fatty acid hydroxylation, deacylation, and phosphate group substitution [22], [23]. These late modifications play a significant role in modulating host responses to infection and the “classical model” with six fatty acids among which 12:0, 14:0 and 14:0(3-OH) chains, constitute an optimal structure for TLR4 recognition and inflammatory response. When fatty acids are removed or modified to shorter or longer derivatives of this structure, the bacteria can escape the receptor signaling system. All Bordetella lipid A structures we have described so far display a common bisphosphorylated β-1,6 glucosamine disaccharide backbone with two amide-linked 14:0(3-OH) substituents [4], [18], [24], [25], [26], [27]. The nature and distribution of ester-linked fatty acids have proved to be species- or strain-specific and highly variable [18], [26]. One of the unusual features of Bordetella lipid A is the absence of symmetry at the C-3 and C-3′ positions. The late-step modifications ability in their biosynthesis allow Bordetellae to escape or alter TLR4-dependent host-defense mechanisms as well as to decrease the ability of antibiotics [21], [22] to cross the bacterial cell wall. It could have contributed to the adaptation of B. petrii, previously isolated from the environment, to diverse niches or hosts, leading to its relatively recent appearance as a human pathogen [3].

The structural characterization of lipid A from both environmental and human B. petrii isolates will help to elucidate evolutionary and host-adaptation mechanisms of the newly emerging human pathogen. In the present study, we report for the first time the presence of 10:0(3-OH) in the lipid A of B. petrii isolated from a Cystic fibrosis (CF) patient. In Bordetellae, a short-chain 10:0(3-OH), fatty acid is present only in the lipid A of B. pertussis and B. parapertussis, the agents of whooping cough in humans. We hypothesise that the presence of 10:0(3-OH), can be considered as a marker for strict human Bordetella isolates and that B. petrii the only environmental bacterium in this group could have adapted as a human pathogen through modification of its LPS structure.