Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria

Bacteria of the phylum Bacteroidetes, including commensal organisms and opportunistic pathogens, harbor abundant surface-exposed multiprotein membrane complexes (Sus-like systems) involved in carbohydrate acquisition. These complexes have been mostly linked to commensalism, and in some instances, they have also been shown to play a role in pathogenesis. Sus-like systems are mainly composed of lipoproteins anchored to the outer membrane and facing the external milieu. This lipoprotein localization is uncommon in most studied Gram-negative bacteria, while it is widespread in Bacteroidetes. Little is known about how these complexes assemble and particularly about how lipoproteins reach the bacterial surface. Here, by bioinformatic analyses, we identify a lipoprotein export signal (LES) at the N termini of surface-exposed lipoproteins of the human pathogen Capnocytophaga canimorsus corresponding to K-(D/E)2 or Q-A-(D/E)2. We show that, when introduced in sialidase SiaC, an intracellular lipoprotein, this signal is sufficient to target the protein to the cell surface. Mutational analysis of the LES in this reporter system showed that the amino acid composition, position of the signal sequence, and global charge are critical for lipoprotein surface transport. These findings were further confirmed by the analysis of the LES of mucinase MucG, a naturally surface-exposed C. canimorsus lipoprotein. Furthermore, we identify a LES in Bacteroides fragilis and Flavobacterium johnsoniae surface lipoproteins that allow C. canimorsus surface protein exposure, thus suggesting that Bacteroidetes share a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane

Structural insights into the mechanism of protein transport by the Type 9 Secretion System translocon

Secretion systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we trap the T9SS of Flavobacterium johnsoniae in the process of substrate transport by disrupting the T9SS motor complex. Cryo-EM analysis of purified substrate-bound T9SS translocons reveals an extended translocon structure in which the previously described translocon core is augmented by a periplasmic structure incorporating the proteins SprE, PorD and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate–carrier protein complex from the translocon is the energy-requiring step in T9SS transport

Evidence for a LOS and a capsular polysaccharide in Capnocytophaga canimorsus

Capnocytophaga canimorsus is a dog's and cat's oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus

New iron acquisition system in Bacteroidetes

Capnocytophaga canimorsus, a dog mouth commensal and a member of the Bacteroidetes phylum, causes rare but often fatal septicemia in humans that have been in contact with a dog. Here, we show that C. canimorsus strains isolated from human infections grow readily in heat-inactivated human serum and that this property depends on a typical polysaccharide utilization locus (PUL), namely, PUL3 in strain Cc5. PUL are a hallmark of Bacteroidetes, and they encode various products, including surface protein complexes that capture and process polysaccharides or glycoproteins. The archetype system is the Bacteroides thetaiotaomicron Sus system, devoted to starch utilization. Unexpectedly, PUL3 conferred the capacity to acquire iron from serotransferrin (STF), and this capacity required each of the seven encoded proteins, indicating that a whole Sus-like machinery is acting as an iron capture system (ICS), a new and unexpected function for Sus-like machinery. No siderophore could be detected in the culture supernatant of C. canimorsus, suggesting that the Sus-like machinery captures iron directly from transferrin, but this could not be formally demonstrated. The seven genes of the ICS were found in the genomes of several opportunistic pathogens from the Capnocytophaga and Prevotella genera, in different isolates of the severe poultry pathogen Riemerella anatipestifer, and in strains of Bacteroides fragilis and Odoribacter splanchnicus isolated from human infections. Thus, this study describes a new type of ICS that evolved in Bacteroidetes from a polysaccharide utilization system and most likely represents an important virulence factor in this group

Biochemical insights into unique Bacteroidetes pore-forming toxins

The human gut microbiota is one of the densest and most complex microbial ecosystems on earth, harbouring hundreds of different species. This high microbial density and species diversity leads to fierce competition for nutrients and space, hence promoting bacterial antagonism. The Gram-negative Bacteroidetes are prominent long-term colonisers of the intestinal flora and represent on average 50% of all bacterial isolates within the human gut. They possess anti-inflammatory, immunomodulatory and metabolic properties and hence play a pivotal role in human health. To outmatch competitors, Bacteroidetes have evolved strategies to directly antagonize opponents through the production of antimicrobial molecules. Among these, the recently discovered Bacteroidales-secreted antimicrobial proteins (BSAPs) are of special interest as they form a novel class of bacterial pore-forming toxins (PFTs). PFTs are produced by a wide range of bacteia and possess the remarkable ability to transition from inert monomeric water-soluble proteins into integral membrane oligomers to form lytic pores. Uniquely, BSAPs possess a Membrane Attack Complex/Perforin (MACPF) domain usually found in eukaryotic pore-forming innate immunity proteins. Incubation of BSAP-sensitive cells with purified BSAPs causes uptake of the membrane-impermeable DNA dye propidium iodide, and co-culture experiments demonstrate that BSAP-producing strains kill sensitive cells in a BSAP-dependent manner. BSAPs are hence the first bacterial MACPF proteins with bactericidal activity. However, the underlying molecular mechanisms of lytic pore formation and receptor recognition remain cryptic. Here, we present first biochemical insights into BSAP receptor recognition and oligomerization, as well as preliminary structural data of a BSAP/receptor complex. Our findings indicate that highly variable BSAP C-terminal domains, located downstream of the MACPF domain, are the sole factor responsible for receptor recognition and binding. Structural characterization of a BSAP/receptor complex further dissects this interaction and how receptor binding affects positioning of the MACPF domain in respect to the membrane plane. Taken together, our data provide first biochemical insights into these novel and poorly characterized PFTs

A bacterial secretion system caught in the act

The T9SS is a novel secretion system exclusively found in Gram-negative bacteria of the phylum Bacteroidetes. It is most notably associated with the human pathogen Porphyromonas gingivalis, the etiologic agent of chronic periodontitis, in which it is responsible for the secretion of the bacterium’s main virulence factors, so-called gingipains. T9SS substrates are secreted in a two-step process, using the general secretory pathway to cross the inner membrane before being translocated across the outer membrane via the T9SS. This second step is mediated by a specific, folded, recognition signal located in the C-terminus of T9SS substrates (the CTD) and requires an inner membrane motor complex powered by the proton motive force. We recently showed that the T9SS OM translocon is formed from the 36-stranded β-barrel protein SprA1. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to the T9SS components PorV and Plug demonstrate that these proteins control access to the lateral opening and to the periplasmic end of the pore, respectively, suggesting an alternating access mechanism in which the two ends of the protein conducting channel are open at different times. This model also suggests that only one conformation of SprA is able to interact with its substrates. We now report our progress in probing this mechanistic model by capturing transport intermediates of the T9SS translocon. We show that removing the T9SS energy source traps substrate proteins during passage through the translocon and allows the isolation of an extended translocon complex (ETC) that contains as additional components the proteins SprE, Fjoh_3466, and a homologue (SkpA) of the Escherichia coli periplasmic chaperone Skp. A structure of the extended translocon shows that the additional translocon components form a disc-shaped structure to one side of the SprA pore at the periplasmic side of the complex. Structural analysis of substrate-translocon complexes isolated by in vivo trapping or in vitro reconstitution reveal that the substrate CTD binds to the extracellular loops of PorV located within the SprA pore. Live cell single molecule tracking experiments imply that the extended translocon is the physiologically relevant form of the transporter. Strikingly, deletion of SprE alone also leads to substrate trapping on the translocon. Our data show that SprE and energization of the T9SS are required for the substrate release step of transport. We propose a model for Type 9 transport in which PorV is used to pull substrate proteins through the translocon

Structure(s) and mechanism of action of a novel bacterial secretion system

The T9SS is a novel secretion system exclusively found in Gram-negative bacteria of the phylum Bacteroidetes1. It is involved in a wide array of processes ranging from biofilm and S-layer formation to secretion of virulence factors in fish and avian pathogens. It is most notably associated with the human pathogen Porphyromonas gingivalis, the etiologic agent of chronic periodontitis, in which it is responsible for the secretion of the bacterium’s main virulence factors, so-called gingipains2. T9SS substrates are secreted in a two-step process, using the general secretory (Sec) pathway to cross the inner membrane before being translocated across the outer membrane (OM) via the T9SS. This second step is mediated by a specific recognition signal located in the C-terminus (the CTD) of T9SS substrates. The CTD is then proteolytically removed and substrates are either released or attached to the cell surface2,3. While all major components of the T9SS have been identified, little was known about the key aspects of the transport process across the OM, including how substrates are recognized and targeted to the secretion pore and, most importantly, the identity of the pore itself. We recently showed that the T9SS translocon is formed from the 36-strand transmembrane β- barrel protein SprA4. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to different T9SS components demonstrate that partner proteins control access to the lateral opening and to the periplasmic end of the pore, suggesting an alternating access mechanism in which the two ends of the protein conducting channel are open at different times. Our recent progress in understanding the molecular mechanism of the T9SS will be described