Volpi et al. demonstrate that hypomorphic EXTL3 mutations cause abnormalities of heparan sulfate composition, affect signaling in response to growth factors and cytokines, and perturb thymopoiesis, resulting in a novel genetic disease associating skeletal dysplasia, T cell immunodeficiency, and neurodevelopmental delay.

These data suggest that alpha-toxin enhances virulence by facilitating the generation of CXC chemokine gradients and stimulating chemokine-induced neutrophil influx in S. aureus pneumonia.

In pneumonia caused by the bacterium Staphylococcus aureus, the intense inflammatory response that is triggered by this infection can lead to the development of lung injury. Little is known, however, about the impact of specific virulence factors on this inflammatory disorder, which causes both significant mortality and morbidity. In this study, we examined the role of ␤-toxin, a neutral sphingomyelinase, in S. aureus-induced lung injury. Our results showed that the central features of lung injury-specifically, increased neutrophilic inflammation, vascular leakage of serum proteins into the lung tissue, and exudation of proteins into the airway-are significantly attenuated in mice infected intranasally with S. aureus deficient in ␤-toxin compared with mice infected with S. aureus expressing ␤-toxin. In addition, intranasal administration of ␤-toxin evoked the characteristic features of lung injury in wild-type mice whereas neutropenic mice were protected from such injury. However, mutant ␤-toxin mice deficient in sphingomyelinase activity failed to trigger features of lung injury. Ablation of sphingomyelinase activity also interfered with the ability of ␤-toxin to stimulate ectodomain shedding of syndecan-1, a major heparan sulfate proteoglycan found in epithelial cells. Moreover, syndecan-1-null mice were significantly protected from ␤-toxin-induced lung injury relative to wild-type mice. These data indicate that S. aureus ␤-toxin is a critical virulence factor that induces neutrophil-mediated lung injury through both its sphingomyelinase activity and syndecan

Many microbial pathogens subvert cell surface heparan sulfate proteoglycans (HSPGs) to infect host cells in vitro. The significance of HSPG-pathogen interactions in vivo, however, remains to be determined. In this study, we examined the role of syndecan-1, a major cell surface HSPG of epithelial cells, in Staphylococcus aureus corneal infection. We found that syndecan-1 null (Sdc1 ؊/؊ ) mice significantly resist S. aureus corneal infection compared with wild type (WT) mice that express abundant syndecan-1 in their corneal epithelium. However, syndecan-1 did not bind to S. aureus, and syndecan-1 was not required for the colonization of cultured corneal epithelial cells by S. aureus, suggesting that syndecan-1 does not mediate S. aureus attachment to corneal tissues in vivo. Instead, S. aureus induced the shedding of syndecan-1 ectodomains from the surface of corneal epithelial cells. Topical administration of purified syndecan-1 ectodomains or heparan sulfate (HS) significantly increased, whereas inhibition of syndecan-1 shedding significantly decreased the bacterial burden in corneal tissues. Furthermore, depletion of neutrophils in the resistant Sdc1 ؊/؊ mice increased the corneal bacterial burden to that of the susceptible WT mice, suggesting that syndecan-1 moderates neutrophils to promote infection. We found that syndecan-1 does not affect the infiltration of neutrophils into the infected cornea but that purified syndecan-1 ectodomain and HS significantly inhibit neutrophil-mediated killing of S. aureus. These data suggest a previously unknown bacterial subversion mechanism where S. aureus exploits the capacity of syndecan-1 ectodomains to inhibit neutrophil-mediated bacterial killing mechanisms in an HS-dependent manner to promote its pathogenesis in the cornea.Microbial pathogens express a multitude of factors that interact with host components. Pathogens use these hostpathogen interactions to their advantage to survive in the host environment. Studies during the last several decades have proposed that many viral, bacterial, and parasitic pathogens bind to cell surface HSPGs 2 to facilitate their initial attachment and subsequent invasion of host cells (1-3). Evidence that the HSPG interaction is biologically important is provided by the finding that HS-binding pathogens show markedly attenuated attachment or invasion of host cells whose HS expression has been reduced by enzymatic treatment or mutagenesis. Furthermore, exogenous HS or heparin (pharmaceutical functional mimic of HS) inhibits pathogen attachment and entry. Furthermore, where examined, mutant strains lacking the HSPG adhesin are viable and show normal growth rates, suggesting that the ability to bind to HSPGs is strictly a virulence activity. However, the significance of HSPG-pathogen interactions in infectious diseases has yet to be clearly established in vivo.Syndecans comprise a major family of cell surface HSPGs (1, 4). Syndecans are type I transmembrane HSPGs composed of four members in mammals. At the cell surface, syndecans function pr...

Several microbial pathogens stimulate the ectodomain shedding of host cell surface proteins to promote their pathogenesis. We reported previously that Pseudomonas aeruginosa and Staphylococcus aureus activate the ectodomain shedding of syndecan-1 and that syndecan-1 shedding promotes P. aeruginosa pathogenesis in mouse models of lung and burned skin infections. However, it remains to be determined whether activation of syndecan-1 shedding is a virulence mechanism broadly used by pathogens. Here we show that Streptococcus pneumoniae stimulates syndecan-1 shedding in cell culture-based assays. S. pneumoniae-induced syndecan-1 shedding was repressed by peptide hydroxamate inhibitors of metalloproteinases but not by inhibitors of intracellular signaling pathways previously found to be essential for syndecan-1 shedding caused by P. aeruginosa, S. aureus, or other shedding agonists. A 170-kDa protein fraction with a peptide hydroxamate-sensitive shedding activity was purified by ammonium sulfate precipitation, DEAE chromatography, and size exclusion chromatography. Mass spectrometry analyses revealed that the 170-kDa fraction is composed of ZmpB and ZmpC, two metalloproteinase virulence factors of S. pneumoniae. Both the purified 170-kDa ZmpB/ ZmpC fraction and unfractionated S. pneumoniae culture supernatant generated syndecan-1 ectodomains that are smaller than those released by endogenous shedding. Further, a mutant S. pneumoniae strain deficient in zmpC, but not zmpB, lost its capacity to stimulate syndecan-1 shedding. These data demonstrate that S. pneumoniae directly sheds syndecan-1 ectodomains through the action of ZmpC.

Background: Syndecan-1 promotes bacterial infections, but how this is accomplished remains unclear. Results: Syndecan-1 and 2-O-sulfated heparan compounds specifically enhanced S. aureus corneal virulence and inhibited bacterial killing by CRAMP secreted from degranulated neutrophils. Conclusion: Specific structural motifs in syndecan-1 HS promote S. aureus corneal infection by inhibiting neutrophil CRAMP. Significance: This study uncovers a new pathogenic role for syndecan-1 in bacterial infection.

Subversion of heparan sulfate proteoglycans (HSPGs) is thought to be a common virulence mechanism shared by many microbial pathogens. The prevailing assumption is that pathogens co-opt HSPGs as cell surface attachment receptors or as inhibitors of innate host defense. However, there are few data that clearly support this idea in vivo. We found that deletion of syndecan-1 (Sdc1), a major cell surface HSPG of epithelial cells, causes a gain of function in a mouse model of scarified corneal infection, where Sdc1−/− corneas were significantly less susceptible to Streptococcus pneumoniae infection. Administration of excess Sdc1 ectodomains significantly inhibited S. pneumoniae corneal infection, suggesting that Sdc1 promotes infection as a cell surface attachment receptor. However, S. pneumoniae did not interact with Sdc1 and Sdc1 was shed upon S. pneumoniae infection, indicating that Sdc1 does not directly support S. pneumoniae adhesion. Instead, Sdc1 promoted S. pneumoniae adhesion by driving the assembly of fibronectin (FN) fibrils in the corneal basement membrane to which S. pneumoniae attaches when infecting injured corneas. S. pneumoniae specifically bound to corneal FN via PavA, and PavA deletion significantly attenuated S. pneumoniae virulence in the cornea. Excess Sdc1 ectodomains inhibited S. pneumoniae corneal infection by binding to the Hep II domain and interfering with S. pneumoniae PavA binding to FN. These findings reveal a previously unknown virulence mechanism of S. pneumoniae where key extracellular matrix (ECM) interactions and structures that are essential for host cell homeostasis are exploited for bacterial pathogenesis. IMPORTANCE Bacterial pathogens have evolved several ingenious mechanisms to subvert host cell biology for their pathogenesis. Bacterial attachment to the host ECM establishes a niche to grow and is considered one of the critical steps of infection. This pathogenic mechanism entails coordinated assembly of the ECM by the host to form the ECM structure and organization that are specifically recognized by bacteria for their adhesion. We serendipitously discovered that epithelial Sdc1 facilitates the assembly of FN fibrils in the corneal basement membrane and that this normal biological function of Sdc1 has detrimental consequences for the host in S. pneumoniae corneal infection. Our studies suggest that bacterial subversion of the host ECM is more complex than previously appreciated.

Heparan sulfate proteoglycans (HSPGs) are at the forefront of host-microbe interactions. Molecular and cell-based studies suggest that HSPG-pathogen interactions promote pathogenesis by facilitating microbial attachment and invasion of host cells. However, the specific identity of HSPGs, precise mechanisms by which HSPGs promote pathogenesis, and the in vivo relevance of HSPG-pathogen interactions remain to be determined. HSPGs also modulate host responses to tissue injury and inflammation, but functions of HSPGs other than facilitating microbial attachment and internalization are understudied in infectious disease.Here we examined the role of syndecan-1 (Sdc1), a major cell surface HSPG of epithelial cells, in mouse models of Listeria monocytogenes (Lm) infection. We show that Sdc1-/mice are significantly less susceptible to both intragastric and intravenous Lm infection compared to wild type (Wt) mice. This phenotype is not seen in Sdc3-/-or Sdc4-/-mice, indicating that ablation of Sdc1 causes a specific gain of function that enables mice to resist listeriosis. However, Sdc1 does not support Lm attachment or invasion of host cells, indicating that Sdc1 does not promote pathogenesis as a cell surface Lm receptor. Instead, Sdc1 inhibits the clearance of Lm before the bacterium gains access to its intracellular niche. Large intravascular aggregates of neutrophils and neutrophil extracellular traps (NETs) embedded with antimicrobial compounds are formed in Sdc1-/-livers, which trap and kill Lm. Lm infection induces Sdc1 shedding from the surface of hepatocytes in Wt livers, which is directly associated with the decrease in size of intravascular aggregated NETs. Furthermore, administration of purified Sdc1 ectodomains or DNase inhibits the formation of intravascular aggregated neutrophils and NETs and significantly increases the liver bacterial burden in Sdc1-/-mice. These data indicate that Lm induces Sdc1 shedding to subvert the activity of Sdc1 ectodomains to inhibit its clearance by intravascular aggregated NETs.