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Staphylococcal protein A (SpA) is anchored to the cell wall envelope of Staphylococcus aureus by sortase A, which links the threonyl (T) of its C-terminal LPXTG motif to peptidoglycan cross-bridges (i.e., Gly5). SpA binds the Fcγ domains of IgG and protects staphylococci from opsonophagocytic clearance. Moreover, SpA cross-links B-cell receptors to modify host adaptive immune responses. The mechanisms whereby SpA is released from the bacterial surface to access the host's immune system are not known. Here we demonstrate that SpA is released with murein tetrapeptide-tetraglycyl [L-Ala-D-iGln-(SpA-Gly5)L-Lys-D-Ala-Gly4] linked to its C-terminal threonyl. LytN, a cross-wall murein hydrolase, contributes to the release of SpA by removing amino sugars [i.e., N-acetylmuramic acid-N-acetylglucosamine (MurNAc-GlcNAc)] from attached peptidoglycan, whereas LytM, a pentaglycyl-endopeptidase, triggers polypeptide release from the bacterial envelope. A model is proposed whereby murein hydrolases cleave the anchor structure of released SpA to modify host immune responses.

The envelope of Staphylococcus aureus is comprised of peptidoglycan and its attached secondary polymers, teichoic acid, capsular polysaccharide, and protein. Peptidoglycan synthesis involves polymerization of lipid II precursors into glycan strands that are cross-linked at wall peptides. It is not clear whether peptidoglycan structure is principally determined during polymerization or whether processive enzymes affect cell wall structure and function, for example, by generating conduits for protein secretion. We show here that S. aureus lacking SagB, a membrane-associated N -acetylglucosaminidase, displays growth and cell-morphological defects caused by the exaggerated length of peptidoglycan strands. SagB cleaves polymerized glycan strands to their physiological length and modulates antibiotic resistance in methicillin-resistant S. aureus (MRSA). Deletion of sagB perturbs protein trafficking into and across the envelope, conferring defects in cell wall anchoring and secretion, as well as aberrant excretion of cytoplasmic proteins. IMPORTANCE Staphylococcus aureus is thought to secrete proteins across the plasma membrane via the Sec pathway; however, protein transport across the cell wall envelope has heretofore not been studied. We report that S. aureus sagB mutants generate elongated peptidoglycan strands and display defects in protein secretion as well as aberrant excretion of cytoplasmic proteins. These results suggest that the thick peptidoglycan layer of staphylococci presents a barrier for protein secretion and that SagB appears to extend the Sec pathway across the cell wall envelope.

Cells of eukaryotic or prokaryotic origin express proteins with LysM domains that associate with the cell wall envelope of bacteria. The molecular properties that enable LysM domains to interact with microbial cell walls are not yet established. Staphylococcus aureus, a spherical microbe, secretes two murein hydrolases with LysM domains, Sle1 and LytN. We show here that the LysM domains of Sle1 and LytN direct murein hydrolases to the staphylococcal envelope in the vicinity of the cross-wall, the mid-cell compartment for peptidoglycan synthesis. LysM domains associate with the repeating disaccharide β-N-acetylmuramic acid, (1→4)-β-N-acetylglucosamine of staphylococcal peptidoglycan. Modification of N-acetylmuramic acid with wall teichoic acid, a ribitol-phosphate polymer tethered to murein linkage units, prevents the LysM domain from binding to peptidoglycan. The localization of LytN and Sle1 to the cross-wall is abolished in staphylococcal tagO mutants, which are defective for wall teichoic acid synthesis. We propose a model whereby the LysM domain ensures septal localization of LytN and Sle1 followed by processive cleavage of peptidoglycan, thereby exposing new LysM binding sites in the cross-wall and separating bacterial cells.

Summary Protozoan parasites represent major public health challenges. Many aspects of their cell biology are distinct from their animal hosts, providing potential therapeutic targets. Toxoplasma gondii is a protozoan parasite that contains a divergent regulator of chromosome condensation 1 (TgRCC1) that is required for virulence and efficient nuclear trafficking. RCC1 proteins function as a guanine exchange factor for Ras-related nuclear protein (Ran), an abundant GTPase responsible for the majority of nucleocytoplasmic transport. Here we show that while there are dramatic differences from well-conserved RCC1 proteins, TgRCC1 associates with chromatin, interacts with Ran and complements a mammalian temperature-sensitive RCC1 mutant cell line. During the investigation of TgRCC1, we observed several unprecedented phenotypes for TgRan, despite a high level of sequence conservation. The cellular distribution of TgRan is found throughout the parasite cell, whereas Ran in late branching eukaryotes is predominantly nuclear. Additionally, T. gondii tolerates at least low-level expression of dominant lethal Ran mutants. Wild type parasites expressing dominant negative TgRan grew similarly to wild type in standard tissue culture conditions, but were attenuated in serum-starved host cells and mice. These growth characteristics paralleled the TgRCC1 mutant and highlight the importance of the nuclear transport pathway for virulence of eukaryotic pathogens.

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis , mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δ lcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δ lcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δ lcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes ( tagB , tarF , or tarJ2 ) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δ lcp mutant was not relieved by tunicamycin treatment or by mutation of tagO , whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δ lcp mutant. We propose a model whereby the S. aureus Δ lcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.

Cell cycle progression for the spherical microbe Staphylococcus aureus requires the coordinated synthesis and remodeling of peptidoglycan. The majority of these rearrangements takes place at the mid-cell, in a compartment designated the cross-wall. Secreted polypeptides endowed with a YSIRK-G/S signal peptide are directly delivered to the cross-wall compartment. One such YSIRK-containing protein is the murein hydrolase LytN. lytN mutations precipitate structural damage to the cross-wall and interfere with staphylococcal growth. Overexpression of lytN also affects growth and triggers rupture of the cross-wall. The lytN phenotype can be reversed by the controlled expression of lytN but not by adding purified LytN to staphylococcal cultures. LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-l-alanine amidase and d-alanyl-glycine endopeptidase. Thus, LytN secretion into the cross-wall promotes peptidoglycan separation and completion of the staphylococcal cell cycle.

Apicomplexa is a phylum within the kingdom Protista that contains some of the most significant threats to public health. One of the members of this phylum, Toxoplasma gondii, is amenable to molecular genetic analyses allowing for the identification of factors critical for colonization and disease. A pathway found to be important for T. gondii pathogenesis is the Ran network of nuclear trafficking. Bioinformatics analysis of apicomplexan genomes shows that while Ran is well conserved, the key regulators of Ran--Regulator of Chromosome Condensation 1 and Ran GTPase activating protein--are either highly divergent or absent. Likewise, several import and export receptor molecules that are crucial for nuclear transport are either not present or have experienced genetic drift such that they are no longer recognizable by bioinformatics tools. In this minireview we describe the basics of nuclear trafficking and compare components within apicomplexans to defined systems in humans and yeast. A detailed analysis of the nuclear trafficking network in these eukaryotes is required to understand how this potentially unique cellular biological pathway contributes to host-parasite interactions.

Eukaryotic parasites are a leading cause of morbidity and mortality worldwide, yet little is known about the genetic basis of their virulence. Here, we present a forward genetic screen to study pathogenesis in the protozoan parasite Toxoplasma gondii . By using modified signature-tagged mutagenesis, the growth of 6,300 T. gondii insertional mutants was compared in cell culture and murine infection to identify genes required specifically in vivo . One of the 39 avirulent mutants is disrupted in a divergent ortholog of the regulator of chromosome condensation 1 (RCC1), which is critical for nuclear trafficking in model systems. Although this RCC1 mutant grows similar to wild type in standard tissue culture conditions, it is growth-impaired under nutrient limitation. Genetic complementation of mutant parasites with the T. gondii RCC1 gene fully restores both virulence in mice and growth under low-nutrient conditions. Further analysis shows that there is a significant defect in nuclear trafficking in the RCC1 mutant. These findings suggest that the rate of nuclear transport is a critical factor affecting growth in low-nutrient conditions in vivo and in vitro . Additionally, we observed that although RCC1 proteins are highly conserved in organisms from humans to yeast, no protozoan parasite encodes a characteristic RCC1. This protein divergence may represent a unique mechanism of nucleocytoplasmic transport. This study illustrates the power of this forward genetics approach to identify atypical virulence mechanisms.