Olaf Schneewind, M.D., Ph.D.

Bacterial Pathogenesis and Protein Targeting

Research Summary

Our laboratory examines the mechanisms and strategies whereby pathogenic bacteria cause human disease. Several different microbes are being investigated.

The cell wall of Staphylococcus aureus and other Gram-positive pathogens can be viewed as a surface organelle with anchored proteins that interact with the host environment during infection. Our research has revealed four different mechanisms of protein anchoring to the cell wall envelope. Surface proteins bearing a C-terminal sorting signal with a LPXTG motif are cleaved by the sortase A enzyme and linked to the cell wall crossbridges of peptidoglycan. This group of surface proteins is essential for pathogenesis and mediates bacterial attachment to host tissues and escape from the immune system. Surface proteins bearing a C-terminal sorting signal with a NPQTN motif are cleaved by sortase B. This mechanism is involved in iron transport during infection and is required for bacterial persistence in the host. Autolysins are enzymes that cleave the cell wall envelope at defined sites. One group of autolysins, e.g. lysostaphin and f11 hydrolase, is targeted to a receptor that is distributed uniformly over the bacterial surface. Another autolysins is targeted to the equatorial surface rings of staphylococci and mediates peptidoglycan cleavage at cell divisions sites. Our laboratory entertains genetic, molecular biological, biochemical, microscopic as well as animal infectious strategies to reveal mechanisms of protein targeting and the role in the establishment of disease. Our results are useful for the design of new therapies that can be used for the treatment of human infections caused by S. aureus and other Gram-positive bacteria.

Pathogenic Yersinia spp. invade their human hosts and colonize lymphoid tissues. This unique infectious strategy requires bacterial mechanisms of immune evasion. Yersinia type III secretion prevents the phagocytic killing of bacteria during infection. Further, the type III machinery intoxicates and kills immune cells, thereby impairing the host's ability to clear invasive Yersinia. In fact, the pathogenesis of the most notorious of all pathogens, Yersinia pestis - the causative agent of plague, relies on the type III secretion machinery. We are interested in the mechanisms of protein recognition and transport by the type III machinery. Our results suggest that mRNAs of Yops, the substrates of the type III machinery, harbor signals that lead to the secretion of the encoded polypeptide chains. Current work is mapping the secretion signals and analyzing the mode of mRNA recognition by the type III machinery. A second area of research is the regulation of the type III secretion machinery. Upon bacterial entry into the host, Yersinia sense three environmental signals: a temperature shift to 37 ºC, glutamate ions as well as serum proteins. These signals trigger yersiniae to express and assemble the type III machinery and to transport YopB, YopD, YopR and LcrV into the extra-cellular milieu. Docking of yersiniae on the surface of immune cells leads to the insertion of type III secretion machinery needles into the plasma membrane of host cells and in sensing of the low calcium concentration of the cell's interior. Yersinia respond to the low calcium signal by transporting YopE, YopH, YopM, YopN, YopO, YopP, YopT and YscM (LcrQ) into the cytosol of host cells. The sum of the function of these toxic proteins leads to a block in phagocytosis and in the killing of macrophages. Research on type III secretion represents a hotly contested frontier of microbiological science. Our results will be useful for the treatment of human infections caused by Yersinia, including the biological warfare agent Yersinia pestis, as well as several other Gram-negative pathogens.

Selected Papers

Mazmanian, S.K., Skaar, E., Gaspar, A., Humayun, M., Jelesnka, J., Gornicki, P., Joachimiak, A., Missiakas, D. & Schneewind, O. (2003). Passage of heme-iron across the envelope of Staphylococcus aureus. Science 299: 906-909.

Cambronne ED, Sorg JA, Schneewind O. (2004). Binding of SycH chaperone to YscM1 and YscM2 activates effector yop expression in Yersinia enterocolitica. J Bacteriol. 186: 829-841.

Skaar EP, Schneewind O. (2004). Iron-regulated surface determinants (Isd) of Staphylococcus aureus: stealing iron from heme. Microbes Infect. 6: 390-397.

Ton-That H, Schneewind O. (2004). Assembly of pili in Gram-positive bacteria. Trends Microbiol.12: 228-234.

Marraffini LA, Ton-That H, Zong Y, Narayana SV, Schneewind O. (2004). Anchoring of surface proteins to the cell wall of Staphylococcus aureus. A conserved arginine residue is required for efficient catalysis of sortase A. J Biol Chem. 279: 37763-37770.

Bae, T., Banger, A.K., Wallace, A., Glass, E.M., Aslund, F., Schneewind, O., Missiakas, D.M. (2004). Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing. Proc. Natl. Acad. Sci. USA 101:12312-12317.

Goss J.W., Sorg J.A., Ramamurthi K.S., Ton-That H., Schneewind O. (2004). The secretion signal of YopN, a regulatory protein of the Yersinia enterocolitica type III secretion pathway. J Bacteriol. 186: 6320-6324.

Skaar, E.P., Humanyun, M., Bae, T., DeBord, K., Schneewind, O. (2004). Iron source preference of Staphylococcus aureus infections. Science. 10:1626-1628.

Cambronne ED and Schneewind O. (2005). Bacterial invasions: molecular systems dedicated to the invasion of host tissues. Contrib Microbiol 12:181-209.

Wu R, Skaar EP, Zhang R, Joachimiak G, Gornicki P, Schneewind O an dJoachimiak A. (2005). Staphylococcus aureus IsdG and lsdl, heme-degrading enzymes with structural similarity to monooxygenases. J Biol Chem 280:2840-2846.

Ramamurthi KS and Schneewind O. (2005). A synonymous mutation in Yersinia enterocolitica yopE affects the function of the YopE type III secretion signal. J Bacteriol 187:707-715.

Marraffini LA and Schneewind O. (2005). Anchor structure of staphylococcal surface proteins. V. Anchor structure of the sortase GB substrate IsdC. J Biol Chem 280:16263-16271.

Mattner J, Debord KL, Ismail N, Goff RD, Cantu C 3rd, Zhou D, Saint-Mezard P, Wang V, Gao Y, Yin N, Hoeve K, Schneewind O, Walker D, Beutler B, Teyton L, Savage PB and Bendelac A. (2005). Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434:525-529.

Gaspar AH, Marraffini LA, Glass EM, Debord KL, Ton-That H and Schneewind O. (2005). Bacillus anthracis sortase A (SrtA) anchors LPXTG motif-containing surface proteins to the cell wall envelope. J Bacteriol 187:4646-4655.

Overheim KA, DePaolo RW, DeBord KL, Morrin EM, Anderson DM, Green NM, Brubaker RR, Jabri B and Schneewind O. (2005). LcrV plague vaccine with altered immunomodulatory properties. Infect Immun 73:5152-5159.

Marketon MM, DePaolo RW, DeBord KL, Jabri B and Schneewind O. (2005). Plague bacteria target immune cells during infection. Science 309:1739-1741.

Sorg JA, Miller NC and Schneewind O. (2005). Substrate recognition of type III secretion machines --testing the RNA signal hypothesis. Cell Microbiol 7:1217-1225.

Sorg JA, Miller NC, Marketon MM and Schneewind O. (2005). Rejection of impassable substrates by Yersinia type III secretion machines. J Bacteriol 187:7090-7102.

Blaylock B and Schneewind O. (2005). Microbiology: loading the type III cannon. Nature 437:821.

Bae T and Schneewind O. (2006). Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmic 55:58-63.

Skaar EP, Gaspar AH and Schneewind O. (2006). Bacillus anthracis IsdG, a heme-degrading monooxygenase. J Bacteriol 188:1071-1080.

Marraffini LA, DeDent AC and Schneewind O. (2006). Sortases adn the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol Mol Biol Rev 70::192-221.

Grundling A and Schneewind O. (2006). Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus. J Bacteriol. 188:2463-2472.

Blaylock B, Riordan KE, Missiakas DM and Schneewind O. (2006). Characterization of the Yersinia enterocolitica type III secretion ATPase YscN and its regulator YscL. J Bacteriol 188:3525-3534.

Maresso AW and Schneewind O. (2006). Iron acquisition and transport in Staphylococcus aureus. Biometals 19:193-203.

DeBord KL, Anderson DM, Marketon MM, Overheim KA, DePaolo RW, Ciletti NA, Jabri B and Schneewind O. (2006). Immunogenicity and protective immunity against bubonic plague and pneumonic plague by immunization of mice with the recombinant V10 antigen, a variant of LcrV. Infect Immun 74:4910-4914.

Grundling A, Missiakas DN and Schneewind O. (2006). Staphylococcus aureus mutants with increased lysostaphin resistance. J Bacteriol 188:6286-6297.

Maresso AW, Chapa TJ and Schneewind O. (2006). Surface protein IsdC and sortase B are required for heme-iron scavenging of Bacillus anthracis. J Bacteriol 188(23):8145-8152.

Torres VJ, Pishchany G, Humayun M, Schneewind O and Skaar EP. (2006). Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization. J Bacteriol 188:8421-8429.

Sorg JA, Blaylock B and Schneewind O. (2006). Secretion signal recognition by YscN, the Yersinia type III secretion ATPase. Proc Nat Acad Sci USA 103:16490-16495.

Budzik JM and Schneewind O. (2006). Pili prove pertinent to enterococcal endocarditis. J Clin Inv 16:2582-2584.

Stranger-Jones YK, Bae T and Schneewind O. (2006). Vaccine assembly from surface proteins of Staphylococcus aureus. Proc Nat Acad Sci USA 103:16942-16947.

Marraffini LA and Schneewind O. (2006). Targeting proteins to the cell wall of sporulating Bacillus anthracis. Mol Microbiol 62:1402-1417.

Chen PR, Bae T, Williams WA, Duguid EM, Rice PA, Schneewind O and He C. (2006). An oxidation-sensing mechanism is used by the global regulator MgrA in Staphylococcus aureus. Nat Chem Biol 2:591-595.

Bae T, Baba T, Hiramatsu K and Schneewind O. (2006). Prophages of Staphylococcus aureus Newman and their contribution to virulence. Mol Microbiol 62:1035-1047.

Bubeck-Wardenburg J, Patel R and Schneewind O. (2007). Surface proteins and exotoxins are required for the pathogenesis of Staphylococcus aureus pneumonia. Infect Immun 75:1040-1044.

Grundling A and Schneewind O. (2007). Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus. Proc Natl Acad Sci USA 104:8478-8483.

Maresso AW, Wu R, Kern JW, Zhang R, Janik D, Missiakas DM, Duban ME, Joachimiak A and Schneewind O. (2007). Activation of inhibitors by sortase triggers irreversible modification of the active site. J Biol Chem 282:23129-23139.

Marraffini LA and Schneewind O. (2007). Sortase C-mediated anchoring of Basl to the cell wall envelope of Bacillus antracis. J Bacteriol 189:6425-6436.

Quenee LE and Schneewind O. (2007). Ubiquitin-Yop hybrids as proves for post-translational transport by the Yersinia type III secretion pathway. Mol Microbiol 65:386-400.

Budzik JM, Marrafini LA and Schneewind O. (2007). Assembly of pili on the surface of Bacillus cereus vegetative cells. Mol Microbiol 66:495-510.

Cornelius C, Quenee L, Anderson D and Schneewind O. (2007). Protective immunity against plague. Adv Exp Med Biol 603:415-424.

Fujiya M, Musch MW, Nakagawa Y, Hu S, Alverdy J, Kohgo Y, Schneewind O, Jabri B and Chang EB. (2007). The Bacillus subtilis quorum-sensing molecule CSF contributes to intestinal homeostasis via OCTN2, a host cell membrane transporter. Cell Host Microbe 1:299-308.

Wardenburg JB, Bae T, Otto M, DeLeo FR and Schneewind O. (2007). Poring over pores: alpha-hemolysin and Panto-Valentine leukocidin in Staphylococcus aureus pneumonia. Nat Med 13:405-406.

Baba T, Bae T, Schneewind O, Takeuchi F and Hiramatsu K. (2008). Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands. J Bacteriol 190:300-310.

Kern JW and Schneewind O. (2008). BsIA, a pXO1-encoded adhesin of Bacillus anthracis. Mol Microbiol 68:504-515.

Quenee Le, Cornelius CA, Ciletti NA, Elli D and Schneewind O. (2008). Yersinia pestis caf1 (F1) variants and the limits of plague vaccine protection. Infect Immun E pub. ahead of schedule

Maresso AW and Schneewind O. (2008). Sortase as a target of anti-infective therapy. Pharmacol Rev 60:128-141.

Bubeck Wardenburg J and Schneewind O. (2008). Vaccine protection against Staphylococcus aureus pneumonia. J Exp Med 205:287-294.

Bae T, Glass EM, Schneewind O and Missiakas D. (2008). Generating a Collection of Insertion Mutations in the Staphylococcus aureus genome using bursa aurealis. Methods Mol Biol 416:103-116.