Enteropathogenic Escherichia coli and enterohemorrhagic E. coli are diarrheagenic bacterial human pathogens that cause severe gastroenteritis. These enteric pathotypes, together with the mouse pathogen Citrobacter rodentium, belong to the family of attaching and effacing pathogens that form a distinctive histological lesion in the intestinal epithelium. The virulence of these bacteria depends on a type III secretion system (T3SS), which mediates the translocation of effector proteins from the bacterial cytosol into the infected cells. The core architecture of the T3SS consists of a multi-ring basal body embedded in the bacterial membranes, a periplasmic inner rod, a transmembrane export apparatus in the inner membrane, and cytosolic components including an ATPase complex and the C-ring. In addition, two distinct hollow appendages are assembled on the extracellular face of the basal body creating a channel for protein secretion: an approximately 23 nm needle, and a filament that extends up to 600 nm. This filamentous structure allows these pathogens to get through the host cells mucus barrier. Upon contact with the target cell, a translocation pore is assembled in the host membrane through which the effector proteins are injected. Assembly of the T3SS is strictly regulated to ensure proper timing of substrate secretion. The different type III substrates coexist in the bacterial cytoplasm, and their hierarchical secretion is determined by specialized chaperones in coordination with two molecular switches and the so-called sorting platform. In this review, we present recent advances in the understanding of the T3SS in attaching and effacing pathogens.

Our studies reveal that the oral colonizer and cause of infective endocarditis Streptococcus oralis subsp. dentisani displays a striking monolateral distribution of surface fibrils. Furthermore, our data suggest that these fibrils impact the structure of adherent bacterial chains. Mutagenesis studies indicate that these fibrils are dependent on three serine-rich repeat proteins (SRRPs), here named fibril-associated protein A (FapA), FapB, and FapC, and that each SRRP forms a different fibril with a distinct distribution. SRRPs are a family of bacterial adhesins that have diverse roles in adhesion and that can bind to different receptors through modular nonrepeat region domains. Amino acid sequence and predicted structural similarity searches using the nonrepeat regions suggested that FapA may contribute to interspecies interactions, that FapA and FapB may contribute to intraspecies interactions, and that FapC may contribute to sialic acid binding. We demonstrate that a fapC mutant was significantly reduced in binding to saliva. We confirmed a role for FapC in sialic acid binding by demonstrating that the parental strain was significantly reduced in adhesion upon addition of a recombinantly expressed, sialic acid-specific, carbohydrate binding module, while the fapC mutant was not reduced. However, mutation of a residue previously shown to be essential for sialic acid binding did not decrease bacterial adhesion, leaving the precise mechanism of FapC-mediated adhesion to sialic acid to be defined. We also demonstrate that the presence of any one of the SRRPs is sufficient for efficient biofilm formation. Similar structures were observed on all infective endocarditis isolates examined, suggesting that this distribution is a conserved feature of this S. oralis subspecies.

Type three secretion systems (T3SSs) are virulence determinants employed by several pathogenic bacteria as molecular syringes to inject effector proteins into host cells. Diarrhea‐producing enteropathogenic Escherichia coli (EPEC) uses a T3SS to colonize the intestinal tract. T3S is a highly coordinated process that ensures hierarchical delivery of three classes of substrates: early (inner rod and needle subunits), middle (translocators), and late (effectors). Translocation of effectors is triggered upon host‐cell contact in response to different environmental cues, such as calcium levels. The T3S substrate specificity switch from middle to late substrates in EPEC is regulated by the SepL and SepD proteins, which interact with each other and form a trimeric complex with the chaperone CesL. In this study, we investigated the link between calcium concentration and secretion regulation by the gatekeeper SepL. We found that calcium depletion promotes late substrate secretion in a translocon‐independent manner. Furthermore, the stability, formation, and subcellular localization of the SepL/SepD/CesL regulatory complex were not affected by the absence of calcium. In addition, we demonstrate that SepL interacts in a calcium‐independent manner with the major export gate component EscV, which in turn interacts with both middle and late secretion substrates, providing a docking site for T3S. These results suggest that EscV serves as a binding platform for both the SepL regulatory protein and secreted substrates during the ordered assembly of the T3SS.

The type III secretion system (T3SS) is a supramolecular machine used by many bacterial pathogens to translocate effector proteins directly into the eukaryotic host cell cytoplasm. Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrheal disease in underdeveloped countries. EPEC virulence relies on a T3SS encoded within a chromosomal pathogenicity island known as the locus of enterocyte effacement (LEE). In this work, we pursued the functional characterization of the LEE-encoded protein EscK (previously known as Orf4). We provide evidence indicating that EscK is crucial for efficient T3S and belongs to the SctK (OrgA/YscK/MxiK) protein family, whose members have been implicated in the formation of a sorting platform for secretion of T3S substrates. Bacterial fractionation studies showed that EscK localizes to the inner membrane independently of the presence of any other T3SS component. Combining yeast two-hybrid screening and pulldown assays, we identified an interaction between EscK and the C-ring/sorting platform component EscQ. Site-directed mutagenesis of conserved residues revealed amino acids that are critical for EscK function and for its interaction with EscQ. In addition, we found that T3S substrate overproduction is capable of compensating for the absence of EscK. Overall, our data suggest that EscK is a structural component of the EPEC T3SS sorting platform, playing a central role in the recruitment of T3S substrates for boosting the efficiency of the protein translocation process.IMPORTANCE The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine-rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP- isolates also bind the cryptic receptor β-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target.

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High-density lipoproteins (HDLs) are a group of different subpopulations of sialylated particles that have an essential role in the reverse cholesterol transport (RCT) pathway. Importantly, changes in the protein and lipid composition of HDLs may lead to the formation of particles with reduced atheroprotective properties. Here, we show that Streptococcus pneumoniae pneumolysin (PLY) and neuraminidase A (NanA) impair HDL function by causing chemical and structural modifications of HDLs. The proteomic, lipidomic, cellular, and biochemical analysis revealed that PLY and NanA induce significant changes in sialic acid, protein, and lipid compositions of HDL. The modified HDL particles have reduced cholesterol acceptor potential from activated macrophages, elevated levels of malondialdehyde adducts, and show significantly increased complement activating capacity. These results suggest that accumulation of these modified HDL particles in the arterial intima may present a trigger for complement activation, inflammatory response, and thereby promote atherogenic disease progression.

Bacillus thuringiensis (Bt) produces insecticidal proteins that are active against different insect orders. In particular, the family of the three domain-Cry toxins (3d-Cry) have important applications in controlling insect pests as spray products or in transgenic Bt plants. At least three different models account for the mode of action of these proteins. Two of them are based on the pore formation activity of the toxin and propose that larvae are killed by osmotic shock to their midgut cells. Differences in these models are related to the processes of receptor interactions and membrane insertion. The third model proposes that midgut cells are killed through a signal transduction mechanism that is triggered after the binding of 3d-Cry toxins to a cadherin receptor. In this chapter, we will review these models in light of the different resistance mechanisms to Cry toxins that have been documented so far in several insect species.