Type V secretion: mechanism(s) of autotransport through the bacterial outer membrane

Autotransport in Gram-negative bacteria denotes the ability of surface-localized proteins to cross the outer membrane (OM) autonomously. Autotransporters perform this task with the help of a β-barrel transmembrane domain localized in the OM. Different classes of autotransporters have been investigated in detail in recent years; classical monomeric but also trimeric autotransporters comprise many important bacterial virulence factors. So do the two-partner secretion systems, which are a special case as the transported protein resides on a different polypeptide chain than the transporter. Despite the great interest in these proteins, the exact mechanism of the transport process remains elusive. Moreover, different periplasmic and OM factors have been identified that play a role in the translocation, making the term ‘autotransport’ debatable. In this review, we compile the wealth of details known on the mechanism of single autotransporters from different classes and organisms, and put them into a bigger perspective. We also discuss recently discovered or rediscovered classes of autotransporters

Assisted Secretion of a Trimeric Autotransporter Adhesin from Salmonella

Type Vc secretion systems, also known as Trimeric Autotransporter Adhesins (TAAs) are important virulence factors of Gram-negative bacteria.This subclass of bacterial autotransporters forms obligate homotrimers on the surface of bacterial cells, anchored in the outer membrane by the translocator domain at the C-terminus of the protein, through which the rest of the polypeptide is threaded during their biogenesis. During our investigation of the Salmonella adhesin SadA we have discovered that it forms an operon with a small predicted lipoprotein, which we named SadB. As autotransporters are not typically associated with lipoproteins, I was interested in the function of SadB and its interactions with SadA. In this work, I confirmed the prediction that SadB is indeed a lipoprotein, attached to the periplasmic side of the inner membrane. I was able to demonstrate that co-expression of SadB with SadA leads to increased quantity of SadA on the bacterial cell surface, as well as improved folding of the adhesin, which is evidenced by increased protease resistance, as compared to the expression of SadA alone. Additionally, I was able to purify a soluble variant of the protein and obtain a crystal structure with a resolution of 2.45Å. The crystal structure shows, that SadB forms a homotrimer, just as SadA

Complete fiber structures of complex trimeric autotransporter adhesins conserved in enterobacteria

Trimeric autotransporter adhesins (TAAs) are modular, highly repetitive surface proteins that mediate adhesion to host cells in a broad range of Gram-negative pathogens. Although their sizes may differ by more than one order of magnitude, they all follow the same basic head-stalk-anchor architecture, where the head mediates adhesion and autoagglutination, the stalk projects the head from the bacterial surface, and the anchor provides the export function and attaches the adhesin to the bacterial outer membrane after export is complete. In complex adhesins, head and stalk domains may alternate several times before the anchor is reached. Despite extensive sequence divergence, the structures of TAA domains are highly constrained, due to the tight interleaving of their constituent polypeptide chains. We have therefore taken a “domain dictionary” approach to characterize representatives for each domain type by X-ray crystallography and use these structures to reconstruct complete TAA fibers. With SadA from Salmonella enterica, EhaG from enteropathogenic Escherichia coli (EHEC), and UpaG from uropathogenic E. coli (UPEC), we present three representative structures of a complex adhesin that occur in a conserved genomic context in Enterobacteria and is essential in the infection process of uropathogenic E. coli. Our work proves the applicability of the dictionary approach to understanding the structure of a class of proteins that are otherwise poorly tractable by high-resolution methods and provides a basis for the rapid and detailed annotation of newly identified TAAs

A Trimeric Lipoprotein Assists in Trimeric Autotransporter Biogenesis in Enterobacteria

Trimeric autotransporter adhesins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. TAAs form fibrous, adhesive structures on the bacterial cell surface. Their N-terminal extracellular domains are exported through a C-terminal membrane pore; the insertion of the pore domain into the bacterial outer membrane follows the rules of β-barrel transmembrane protein biogenesis and is dependent on the essential Bam complex. We have recently described the full fiber structure of SadA, a TAA of unknown function in Salmonella and other enterobacteria. In this work, we describe the structure and function of SadB, a small inner membrane lipoprotein. The sadB gene is located in an operon with sadA; orthologous operons are only found in enterobacteria, whereas other TAAs are not typically associated with lipoproteins. Strikingly, SadB is also a trimer, and its co-expression with SadA has a direct influence on SadA structural integrity. This is the first report of a specific export factor of a TAA, suggesting that at least in some cases TAA autotransport is assisted by additional periplasmic proteins

Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from <i>Staphylococcus aureus</i>

<div><p>The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In <i>Staphylococcus aureus</i>, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, Rel_<i>Sau</i> or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of Rel_<i>Sau</i> is crucial for the adaption of <i>S</i>. <i>aureus</i> to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of Rel_<i>Sau</i>. Growth pattern, transcriptional analyses and <i>in vitro</i> assays yielded the following results: i) <i>in vivo</i>, under relaxed conditions, as well as <i>in vitro</i>, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) Rel_<i>Sau</i> lacking the CTD exhibits net hydrolase activity when expressed in <i>S</i>. <i>aureus</i> but net (p)ppGpp synthetase activity when expressed in <i>E</i>. <i>coli</i>; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, Rel_<i>Sau</i> primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.</p></div

Influence of the CTD motifs on Rel_<i>Sau</i> interactions.

<p><i>S</i>. <i>aureus</i> (p)ppGpp⁰ was complemented with native and mutated Rel_<i>Sau</i>. Cell lysates were mixed with magnetic beads, coated with Rel_<i>Sau</i> antibodies and enriched proteins identified by MS. Proteins significantly (t-test difference > 0.05) less abundant using TGS mutated versus native Rel_<i>Sau</i> as bait are shown in the first column. The effects of the DC or ACT mutation on interaction with these proteins are shown in the second and third column, respectively.</p