An Acquired and Endogenous Glycocalyx Forms a Bidirectional "Don't Eat" and "Don't Eat Me" Barrier to Phagocytosis.

Macrophages continuously survey their environment in search of pathogens or apoptotic corpses or debris. Targets intended for clearance expose ligands that initiate their phagocytosis ("eat me" signals), while others avoid phagocytosis by displaying inhibitory ligands ("don't eat me" signals). We report that such ligands can be obscured by the glycosaminoglycans and glycoproteins that coat pathogenic as well as malignant phagocytic targets. In addition, a reciprocal barrier of self-synthesized or acquired glycocalyx components on the macrophage surface shrouds phagocytic receptors, curtailing their ability to engage particles. The coating layers of macrophages and their targets hinder phagocytosis by both steric and electrostatic means. Their removal by enzymatic means is shown to markedly enhance phagocytic efficiency. In particular, we show that the removal of mucins, which are overexpressed in cancer cells, facilitates their clearance. These results shed light on the physical barriers that modulate phagocytosis, which have been heretofore underappreciated. VIDEO ABSTRACT.

The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism.

Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors.

Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain-containing protein (PumA) of the multi-drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF-κB, a property transferable to non-PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll-like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin-associated protein 1 (UBAP1), a component of the endosomal-sorting complex required for transport I (ESCRT-I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.

Correction: The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility.

[This corrects the article DOI: 10.1371/journal.ppat.1006092.].

The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility.

Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors are essential for initiating and propagating the eukaryotic innate immune signaling cascade. Here, we investigate TirS, a Staphylococcus aureus TIR mimic that is part of a novel bacterial invasion mechanism. Its ectopic expression in eukaryotic cells inhibited TLR signaling, downregulating the NF-kB pathway through inhibition of TLR2, TLR4, TLR5, and TLR9. Skin lesions induced by the S. aureus knockout tirS mutant increased in a mouse model compared with wild-type and restored strains even though the tirS-mutant and wild-type strains did not differ in bacterial load. TirS also was associated with lower neutrophil and macrophage activity, confirming a central role in virulence attenuation through local inflammatory responses. TirS invariably localizes within the staphylococcal chromosomal cassettes (SCC) containing the fusC gene for fusidic acid resistance but not always carrying the mecA gene. Of note, sub-inhibitory concentration of fusidic acid increased tirS expression. Epidemiological studies identified no link between this effector and clinical presentation but showed a selective advantage with a SCCmec element with SCC fusC/tirS. Thus, two key traits determining the success and spread of bacterial infections are linked.

Contribution of the Twin Arginine Translocation system to the exoproteome of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa uses secretion systems to deliver exoproteins into the environment. These exoproteins contribute to bacterial survival, adaptation, and virulence. The Twin arginine translocation (Tat) export system enables the export of folded proteins into the periplasm, some of which can then be further secreted outside the cell. However, the full range of proteins that are conveyed by Tat is unknown, despite the importance of Tat for the adaptability and full virulence of P. aeruginosa. In this work, we explored the P. aeruginosa Tat-dependent exoproteome under phosphate starvation by two-dimensional gel analysis. We identified the major secreted proteins and new Tat-dependent exoproteins. These exoproteins were further analyzed by a combination of in silico analysis, regulation studies, and protein localization. Altogether we reveal that the absence of the Tat system significantly affects the composition of the exoproteome by impairing protein export and affecting gene expression. Notably we discovered three new Tat exoproteins and one novel type II secretion substrate. Our data also allowed the identification of two new start codons highlighting the importance of protein annotation for subcellular predictions. The new exoproteins that we identify may play a significant role in P. aeruginosa pathogenesis, host interaction and niche adaptation.

BtpB, a novel Brucella TIR-containing effector protein with immune modulatory functions.

Several bacterial pathogens have TIR domain-containing proteins that contribute to their pathogenesis. We identified a second TIR-containing protein in Brucella spp. that we have designated BtpB. We show it is a potent inhibitor of TLR signaling, probably via MyD88. BtpB is a novel Brucella effector that is translocated into host cells and interferes with activation of dendritic cells. In vivo mouse studies revealed that BtpB is contributing to virulence and control of local inflammatory responses with relevance in the establishment of chronic brucellosis. Together, our results show that BtpB is a novel Brucella effector that plays a major role in the modulation of host innate immune response during infection.